ELISA detection of urine DEK to predict and diagnose bladder cancer in humans

ABSTRACT

The present invention is directed to a method of detecting a DEK protein in a human urine sample using an ELISA assay. Methods and compositions for detection of DEK using mAb 260-6F9F6 (as detection antibody) and mAb 16-2C9C3 (as capture antibody) in human urine are provided herein. Specifically, the ELISA assay utilizes a capture mAb and a detection mAb to yield a high sensitivity of &lt;50 ng/mL. The presence of DEK in urine is useful in predicting or diagnosing the occurrence of bladder cancer in humans.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. Pat. No. 9,255,925,filed on Apr. 22, 2014, which is a Continuation-In-Part (CIP) of U.S.Pat. No. 8,741,582, filed Oct. 20, 2011, which claims the benefit ofpriority under 35 U.S.C. §119(e) to U.S. Provisional Application Nos.61/455,406 filed Oct. 20, 2010 and 61/455,405 filed Oct. 20, 2010, thedisclosures of which are hereby incorporated by reference in theirentireties.

FIELD OF INVENTION

The present invention generally relates to a method of detecting a DEKprotein in a urine sample. Specifically, the present invention relatesto a method of detecting and diagnosing bladder cancer in humans byELISA to detect DEK in human urine. The ELISA utilizes a firstmonoclonal antibody to capture DEK and a second monoclonal antibody todetect DEK to provide a high sensitivity assay (i.e., limit of detection<50 ng/mL). The present ELISA method permits a quantitative correlationbetween the presence of a DEK protein in urine with occurrence ofbladder cancer in humans.

BACKGROUND OF THE INVENTION

Bladder cancer is a prevalent malignancy in the United States. In 2010,approximately 70,000 newly diagnosed cases of bladder cancer areexpected; of those, more than 14,000 are expected to die. According tothe American Cancer Society, the five-year survival rate for patientsdiagnosed with bladder cancer is 98% at stage 0, 88% at stage I, 63% atstage II, 46% at stage III, and 15% at stage IV. These bleak statisticshighlight the fact that early detection of bladder cancer is criticalfor the intervention of the disease. The estimated overall cost perpatient from diagnosis of bladder cancer to death is about US$96,000-$187,000; and the total cost amounts to US $3.7 billion.

Early detection of bladder cancer is essential for removing the tumorwith preservation of the bladder, avoiding local complications from thetumor such as bleeding or infections, avoiding metastasis and henceimproving prognosis and long-term survival. In bladder cancer, ˜90% aretransitional cell carcinomas, ˜5% are squamous cell carcinomas, and ˜2%are adenocarcinomas. Of the transitional cell carcinomas, ˜75% presentas superficial tumors; of which ˜50-70% will recur and ˜10-20% willprogress to invasive bladder tumors. Patients are therefore kept undersurveillance for early detection of recurrences.

The current standard methods to detect bladder cancer include cystoscopyand urine cytology. Cystoscopy involves inserting a thin, lighted scopethrough the patient's urethra into the bladder. It is invasive,unpleasant, and expensive, which in turn leads to poor patientcompliance. In addition, cystoscopy often yields false-positive results.Urine cytology is an alternative procedure that involves checking thenumber and appearance of cells in a urine sample. It has a lowsensitivity for detecting small or low-grade bladder tumors.

Numerous urine-based markers have been tested for bladder cancerdetection and surveillance. These markers include complement factor H(BTA-Stat/TRAK), nuclear matrix proteins (NMP22), mucin-like antigens,hyaluronic acid, hyaluronidase, survivin, soluble Fas, telomerase anddetection of chromosomal aneuploidy and deletion using fluorescence insitu hybridization (UroVysion®). However, none have acceptablesensitivity and specificity as a routine tool for bladder cancerdiagnostics and surveillance.

Accordingly, there remains a continuing need for a urine-based test withadequate sensitivity and specificity in the detection and diagnosis ofbladder cancer in humans. It would be advantageous to develop anon-invasive and reliable screening method that encourages initial andfollow-up screening. The present invention cures all the prior artdeficiencies and provides a novel method of detecting DEK protein inurine. The present method provides a high sensitivity and specificity,and can be used as a diagnostic tool to detect bladder cancer in humans.

SUMMARY OF INVENTION

In one aspect, the present invention provides a method of detecting DEKin a urine sample of a human, comprising the steps of: (a) forming aprecipitate from a urine sample with a chemical compound selected fromthe group consisting of acetone, trichloroacetic acid, ethanol,methanol/chloroform, and ammonium sulfate; (b) re-suspending saidprecipitate in a polar solvent to form a solution, said solution has afinal volume that is 10-50 fold less than that of said urine sample; (c)concentrating said solution 2-10 fold by filtration; and (d) detectingDEK in said concentrated solution using an anti-DEK antibody in aWestern blot assay.

Preferably, the chemical compound is acetone, methanol/chloroform, ortrichloroacetic acid, or acetone. Preferably, the chemical compound andurine sample has a volume to volume ratio of 10:1. More preferably, thechemical compound and urine sample has a volume to volume ratio of 5:1or 2:1.

Preferably, the polar solvent is tri-ethanol amine. Preferably, thesolution in step (b) has a final volume of 15-40 fold less than that ofurine sample. Preferably, the solution in step (b) has a final volume of20 fold less than that of urine sample. More preferably, theconcentrated solution in step (c) has a final volume of 5 fold less thanthat of re-suspended solution.

Preferably, the filtration is performed using a filter that has 3 kDcutoff. The anti-DEK antibody is a monoclonal antibody or a polyclonalantibody. The anti-DEK protein is labeled with horse radish peroxidase.

In another aspect, the present invention provides a method of detectingbladder cancer in a human, comprising the steps of: (a) obtaining aurine sample from a human; (b) forming a precipitate from said urinesample with a chemical compound selected from the group consisting ofacetone, trichloroacetic acid, ethanol and ammonium sulfate; (c)re-suspending said precipitate in a polar solvent to form a solution,said solution has a final volume that is 10-50 fold less than that ofurine sample; (d) concentrating said solution 2-10 fold by filtration;and (e) detecting DEK in said concentrated solution using an anti-DEKantibody in a Western blot assay, wherein the presence of DEK protein insaid urine sample is an indicative of a bladder cancer in said human.

Preferably, the bladder cancer is a transitional cell carcinoma.

In yet another aspect, the present invention provides a kit fordetecting bladder cancer in a human, comprising: (a) a container for aurine sample; (b) a chemical compound, wherein said chemical compoundinduces the formation of a precipitate from said urine sample; (c) apolar solvent; (d) a filter with a 3 kD cutoff; and (e) an instructionfor the use of said chemical and said filter in preparing said urinesample to allow detection of DEK protein by Western blot assay.

In one aspect, the present invention provides a method of detecting DEKisoform 2 protein in a urine sample of a human, comprising the steps of:(a) concentrating a urine sample, said urine sample is suspected ofcontaining DEK isoform 2 protein; (b) immobilizing said concentratedurine sample onto a solid surface; (c) adding an anti-DEK antibody tosaid immobilized concentrated urine sample so as to allow a complexformation between DEK isoform 2 protein and said anti-DEK antibody,wherein said anti-DEK antibody recognizes DEK isoform 2 protein; and (d)detecting said protein-antibody complex.

Preferably, the concentrating step is performed by filtration-inducedconcentration of urine. Preferably, the concentrated urine sample is atleast 20 fold concentrated as compared to neat urine. More preferably,the concentrated urine sample is at least 30 fold concentrated ascompared to neat urine.

Preferably, the anti-DEK antibody is a monoclonal antibody or apolyclonal antibody. Preferably, the anti-DEK protein is labeled withhorse-radish peroxidase.

In another aspect, the present invention provides a method of detectingbladder cancer in a human, comprising the steps of: (a) obtaining aurine sample from a human suspected of suffering from bladder cancer;(b) concentrating said urine sample; (c) immobilizing said concentratedurine sample onto a solid surface; (d) adding an anti-DEK antibody tosaid immobilized concentrated urine sample so as to allow a complexformation between DEK isoform 2 protein and said anti-DEK antibody,wherein said anti-DEK antibody recognizes DEK isoform 2 protein; and (e)detecting said protein-antibody complex, wherein the presence of saidprotein-antibody complex is indicative of a bladder cancer in saidhuman.

In yet another aspect, the present invention provides a method ofdetecting DEK isoform 2 protein in a urine sample of a human, comprisingthe steps of: (a) concentrating a urine sample, said urine sample issuspected of containing DEK isoform 2 protein; (b) immobilizing a firstanti-DEK antibody onto a solid surface; (c) adding said concentratedurine sample onto said solid surface having said immobilized firstanti-DEK antibody and allowing formation of DEK isoform 2 protein andfirst anti-DEK antibody complex; (d) removing unbound DEK isoform 2protein; (e) adding a second anti-DEK antibody so as to allow formationof a complex between said bound DEK isoform 2 protein with said secondanti-DEK antibody; and (f) detecting said bound DEK isoform 2 proteinwith said second anti-DEK antibody complex, wherein said first anti-DEKantibody and said second anti-DEK antibody recognize a different regionof DEK isoform 2 protein.

In another aspect, the present invention provides a method of detectingbladder cancer in a human, comprising the steps of: (a) obtaining aurine sample from a human suspected of suffering from bladder cancer;(b) concentrating said urine sample; (c) immobilizing a first anti-DEKantibody onto a solid surface; (d) adding said concentrated urine sampleonto said solid surface having said immobilized first anti-DEK antibodyand allowing formation of DEK isoform 2 protein and first anti-DEKantibody complex; (e) removing unbound DEK isoform 2 protein; (f) addinga second anti-DEK antibody so as to allow formation of a complex betweensaid bound DEK isoform 2 protein with said second anti-DEK antibody; and(g) detecting said bound DEK isoform 2 protein with said second anti-DEKantibody complex, wherein said first anti-DEK antibody and said secondanti-DEK antibody recognize a different region of DEK isoform 2 protein,and wherein the presence of said protein-antibody complex is indicativeof a bladder cancer in said human.

In yet another aspect, the present invention provides a kit fordetecting bladder cancer in a human, comprising: (a) a container for aurine sample; (b) anti-DEK antibody, the anti-DEK antibody recognizesDEK isoform 2 protein; (c) an instruction for the use of said antibodyin detecting DEK isoform 2 protein in an ELISA.

Preferably, the kit further comprises a microtiter plate. Preferably,the kit further comprises a detection reagent. Preferably, the kitfurther comprises an additional anti-DEK antibody, the additionalanti-DEK-antibody also recognizes DEK isoform 2, but with a recognitionsite differs from that of said anti-DEK antibody.

In yet another aspect, the present invention provides a method ofdetecting DEK isoform 2 protein in a urine sample of a human, comprisingthe steps of: (a) providing a neat urine sample, said urine sample issuspected of containing DEK isoform 2 protein; (b) immobilizing a firstanti-DEK monoclonal antibody onto a solid surface; (c) adding said neaturine sample onto said solid surface having said immobilized firstanti-DEK monoclonal antibody and allowing formation of DEK isoform 2protein and first anti-DEK antibody complex; (d) removing unbound DEKisoform 2 protein; (e) adding a second anti-DEK monoclonal antibody soas to allow formation of a complex between said bound DEK isoform 2protein with said second anti-DEK monoclonal antibody; and (f) detectingsaid bound DEK isoform 2 protein with said second anti-DEK monoclonalantibody complex, wherein said first anti-DEK monoclonal antibody andsaid second anti-DEK monoclonal antibody recognize a different region ofDEK isoform 2 protein (SEQ ID NO: 2)

In another aspect, the present invention provides a method of detectingbladder cancer in a human, comprising the steps of: (a) obtaining aurine sample from a human suspected of suffering from bladder cancer;(b) immobilizing a first anti-DEK monoclonal antibody onto a solidsurface; (d) adding said urine sample onto said solid surface havingsaid immobilized first anti-DEK monoclonal antibody and allowingformation of DEK isoform 2 protein and first anti-DEK monoclonalantibody complex; (e) removing unbound DEK isoform 2 protein; (f) addinga second anti-DEK monoclonal antibody so as to allow formation of acomplex between said bound DEK isoform 2 protein with said secondanti-DEK monoclonal antibody; and (g) detecting said bound DEK isoform 2protein with said second anti-DEK antibody complex, wherein said firstanti-DEK monoclonal antibody and said second anti-DEK monoclonalantibody recognize a different region of DEK isoform 2 protein, andwherein the presence of said protein-antibody complex is indicative of abladder cancer in said human.

In one aspect, the present invention provides an isolated monoclonalantibody (mAb 16-2C9C3), said mAb 16-2C9C3 comprises: (i) CDR1 of theheavy chain variable region which has the amino acid sequence of SEQ IDNO:15; (ii) CDR2 of the heavy chain variable region which has the aminoacid sequence of SEQ ID NO:16; (iii) CDR3 of the heavy chain variableregion which has the amino acid sequence of SEQ ID NO:17; (iv) CDR1 ofthe light chain variable region which has an amino acid sequence of SEQID NO: 23; (v) CDR2 of the light chain variable region which has theamino acid sequence of SEQ ID NO:24; and (vi) CDR3 of the light chainvariable region which has the amino acid sequence of SEQ ID NO:25.

In another aspect, the present invention provides an isolated monoclonalantibody (mAb 260-6F9F6), said mAb 260-6F9F6 comprises: (i) CDR1 of theheavy chain variable region which has the amino acid sequence of SEQ IDNO:31; (ii) CDR2 of the heavy chain variable region which has the aminoacid sequence of SEQ ID NO:32; (iii) CDR3 of the heavy chain variableregion which has the amino acid sequence of SEQ ID NO:33; (iv) CDR1 ofthe light chain variable region which has an amino acid sequence of SEQID NO: 39; (v) CDR2 of the light chain variable region which has theamino acid sequence of SEQ ID NO:40; and (vi) CDR3 of the light chainvariable region which has the amino acid sequence of SEQ ID NO:41.

In another aspect, the present invention provides a kit for detectingbladder cancer in a human, comprising: (a) the isolated monoclonalantibody of mAb 16-2C9C3; and (b) an instruction for the use of saidmonoclonal antibody in detecting DEK protein present in urine in anELISA. Preferably, the kit further comprises the isolated monoclonalantibody of mAb 260-6F9F6. The kit may further comprises a microtiterplate, or a detection reagent. Preferably, the kit contains mAb 16-2C9C3and mAb 260-6F9F6. The instruction provides guidance for using (i) mAb16-2C9C3 as a capture antibody, and (ii) mAb 260-6F9F6 as a detectionantibody, for the purpose of binding DEK protein present in a urinesample to said capture antibody to form an immunological complex and(iii) detecting the formation of said immunological complex, such thatthe presence or absence of the immunological complex is indicative ofthe presence or absence of bladder cancer in a human.

In yet another aspect, the present invention provides a method ofdetecting bladder cancer in a human, comprising the steps of: (a)providing a urine sample (neat urine) from a human suspected ofsuffering from bladder cancer; (b) immobilizing the antibody mAb16-2C9C3 onto a solid surface; (c) adding said urine sample onto saidsolid surface to allow DEK protein present in said urine to be capturedonto said solid surface; (d) washing the solid surface to remove unboundDEK protein; (e) adding the antibody mAb 260-6F9F6 so as to allowformation of a complex between said captured DEK protein with saidantibody mAb 260-6F9F6; and (f) detecting said complex, wherein thepresence of said complex is indicative of the presence of DEK in saidurine and whereby detect the occurrence of bladder cancer in said human.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the expression of DEK protein in undifferentiated bladderepithelial cell line (i.e., HBEP), differentiated HBEP, a transformedepithelial cell line (i.e., UroTSA), and four (4) bladder cancer celllines (i.e., RT-4, 5637, T-24 and TCCSUP) in a Western blot assay.β-actin serves as a positive control protein.

FIG. 2 depicts the expression of DEK protein in the cultured media fromfive (5) different cultured cell lines in a Western blot assay. Theseinclude: (i) a transformed epithelial cell line (i.e., UroTSA)transfected with DEK-V5; (ii) UroTSA hosting shRNA against DEK (i.e.,DEK knockdown); (iii) bladder cancer cell line 5637; (iv) bladder cancercell line T-24; and (v) UroTSA. Whole cell lysates from UroTSA (i.e., WClysates of UroTSA) serves as a positive control.

FIG. 3 depicts the expression of DEK protein of bladder tumor tissues(i.e., T) from ten (10) bladder cancer patients in a Western blot assay.Adjacent tissues (i.e., N) from the same patients were used as acomparison. UroTSA serves as a control cell line. β-actin serves as apositive control protein.

FIG. 4 depicts a graphic representation indicating the two (2) DEKisoforms. Note that while DEK isoform 1 is composed of 375 amino acidresidues, DEK isoform 2 lacks the amino acid residues 49-82. Themonoclonal antibody (cat no. 610948) used in this study recognizes onlyDEK isoform 1, whilst the polyclonal antibody (cat no. A301-335A) canrecognize both DEK isoform 1 and DEK isoform 2.

FIG. 5 depicts the expression of DEK protein in urine pellets obtainedfrom four (4) bladder cancer patients (i.e., TCC) and a healthyindividual in a Western blot assay. UroTSA serves as a control.

FIG. 6 depicts a Coomassie-blue stained 10% SDS-PAGE gel of proteinsresolved from urine pellet lysates from four (4) bladder cancer patients(i.e., TCC) as well as a healthy individual.

FIG. 7 depicts the expression of DEK protein in urine supernatant (neat)in a Western blot assay obtained from two (2) bladder cancer patients(i.e., TCC) and two (2) healthy individuals. Bladder cancer cell lineT-24 serves as a control.

FIG. 8 depicts Coomassie-blue stained 10% SDS-PAGE gel of proteinsresolved from the urine supernatant (neat) from two (2) bladder cancerpatients (i.e., TCC), two (2) healthy individuals and bladder cancercell T-24.

FIG. 9 depicts the expression of DEK protein in urine samples that wereconcentrated using a 3 kD filter as detected in a Western blot assay.Urine samples were obtained from one (1) patient with bladder cancer(i.e., TCC), one (1) patient with a history of prostate cancer (i.e.,HxCAP), and one (1) patient with a history of renal cell carcinoma(i.e., HxRCC). UroTSA cell lysate was used as a control.

FIG. 10 depicts the expression of DEK protein in urine samples that weretreated with acetone to obtain precipitates. The re-suspendedprecipitates were subjected to a Western blot assay to detect DEKprotein. Urine samples were obtained from one (1) patient with renalcell carcinoma (i.e., RCC), one (1) patient with prostate cancer (i.e.,CAP), one (1) patient with bladder cancer (i.e., TCC), and one (1)patient with a benign enlarged prostate (i.e., BPH). UroTSA cell lysatewas used as a control.

FIG. 11 depicts Coomassie-blue stained 10% SDS-PAGE gel of the proteinsresolved from acetone precipitated urines from one (1) patient withrenal cell carcinoma (i.e., RCC), one (1) patient with prostate cancer(i.e., CAP), one (1) patient with bladder cancer (i.e., TCC), one (1)patient with benign enlarged prostate (i.e., BPH) and UroTSA celllysates.

FIG. 12 depicts the expression of DEK protein as detected by Westernblot assay in urine samples that were treated with acetone to obtainprecipitates. Urine samples were obtained from four (4) bladder cancerpatients (i.e., TCC). Samples were tested after a single proteinprecipitation with acetone (i.e., single acetone ppt.) and after asecond protein precipitation with acetone (i.e., double acetone ppt.).

FIG. 13 depicts Coomassie-blue stained 10% SDS-PAGE gel of the proteinsresolved from urine samples subjected to single (i.e., Single acetoneppt.) and double acetone precipitation (i.e., Double acetone ppt.).Urine samples were obtained from four (4) bladder cancer patients (i.e.,TCC).

FIG. 14 depicts the expression of DEK protein in urine treated first byfiltration with a 30 kD cut-off membrane filter followed by acetoneprecipitation as detected by Western blot assay. Urine was obtained fromtwo (2) patients suffering from bladder cancer (i.e., TCC). UroTSA celllysate was used as a control.

FIG. 15 depicts Coomassie-blue stained 10% SDS-PAGE gel of the proteinsresolved from urine treated by filtration with a 30 kD cut-off membranefilter followed by acetone precipitation. Samples from two (2) patientssuffering from bladder cancer (i.e., TCC) and UroTSA cell lysate weretested.

FIG. 16 depicts the expression of DEK protein in urine treated first byfiltration with a 30 kD cut-off membrane filter followed by consecutivetriple (3×) acetone precipitations. Urine was obtained from one (1)patient suffering from bladder cancer (i.e., TCC). Expression of DEKprotein in these urines was tested by Western blot assay. UroTSA celllysate was used as a control.

FIG. 17 depicts the expression of DEK protein in urine concentratedfirst by acetone precipitation and then filtered with a 3 kD cut-offmembrane filter as detected in a Western blot assay. Urine was obtainedfrom six (6) patients suffering from prostate cancer (i.e., CAP), five(5) patients suffering from renal cell carcinoma (i.e., RCC), seven (7)patients suffering from transitional cell carcinoma (i.e., TCC) and one(1) patient with a history of transitional cell carcinoma (H.TCC).UroTSA cell lysate was used as a control.

FIG. 18 depicts the number of urine samples per patient groups (i.e.,TCC, CAP, RCC, NMD, and HTCC) that tested positive for DEK in urinesamples by a Western blot assay.

FIG. 19 depicts the recombinant expression of DEK isoform 1 protein asV-5 tagged protein. Total proteins were stained by Coomassie blue andDEK isoform 1 detection was performed by Western Blot using an V-5antibody

FIG. 20 depicts the ELISA sandwich using two (2) different anti-humanDEK antibodies. A rabbit polyclonal anti-human DEK antibody (used ascapture antibody) was coated on the solid support (e.g., microtiterplate) to capture DEK protein. The capture antibody specificallycaptured the DEK protein in spiked urine sample (i.e., recombinant DEKisoform 1 protein added to control urine sample) on the ELISA plate. Thecaptured DEK protein was then detected by a mouse monoclonal anti-DEKantibody. The antibody-antigen sandwich was detected by anti-mouse IgGconjugated with horseradish peroxidase (HRP). The horseradish peroxidaseactivity (representing the level of DEK) was measured by addition oftetramethylbenzidine (TMB) substrate. The color intensity was in directproportion to the amount of the DEK protein. Color development wasstopped and the intensity of the color was measured at optical density(OD) 450 nm on a microtiter plate reader.

FIG. 21 depicts the validation of a developed ELISA using 50 μg oftissue lysates prepared from bladder tumor tissue and adjacent normaltissue.

FIG. 22 depicts a failed experiment in detection of urine DEK from TCCpatients by capture ELISA. Urine samples were subjected to acetoneprecipitation followed by Microcon® concentration from TCC patients. Theurine samples from these TCC patients have been tested positive for DEKexpression by Western blot. Noted that the same urine samples, whenevaluated for DEK presence using capture ELISA, failed to show any DEKprotein.

FIG. 23 depicts urine that subject to acetone precipitation fails to bedetected for DEK by capture ELISA. In this study, recombinant DEKisoform 1 protein was spiked in control urine samples followed byacetone precipitation and re-suspension of precipitates in sucrosebuffer. The re-suspended solution was analyzed by capture DEK ELISA.Noted that recombinant DEK was not detected in the spiked urine samples.In contrast, DEK spiked in control urine (without subject to acetoneprecipitation) could be readily detected by capture ELISA.

FIG. 24 depicts filtration-concentrated urine samples (by Microcon® 3Kfilter to 20 fold concentrated), when assayed by capture DEK ELISA,could not detect DEK.

FIG. 25 depicts an indirect ELISA experiment. Filtration-concentratedurine (by Microcon® 3K filter to 20 fold concentrated) was first coatedthe wells of the microtiter plate. A DEK polyclonal antibody was usedfor DEK detection. Antigen-antibody complex was detected anti-rabbit IgGHRP. The peroxidase activity (representing the level of DEK) wasmeasured by addition of tetramethylbenzidine (TMB) substrate.

FIG. 26 depicts the analysis of 25 healthy donors to determine thecut-off value for the indirect DEK ELISA. Based on the standard curve ofurine spiked with DEK, only two (2) healthy samples out of 25 hadconcentration above 0 μg but less than 0.98 μg. Thus, 1.5 μg was decidedas the cut-off for detection of DEK protein based on standard deviationof all samples tested.

FIG. 27 depicts the detection of DEK in urines of bladder cancerpatients using indirect DEK ELISA. 2 ml of urine sample was concentratedto 100 μl using 3K Microcon® filter to achieve and 20 fold concentrationand analyzed by indirect DEK ELISA. The assay detects DEK protein inmost of the bladder cancer patients with a sensitivity of 65% andspecificity of 89%.

FIG. 28 depicts a schematic representation of the location of thedifferent peptides sequences on the DEK isoform used for the generationof DEK monoclonal antibodies.

FIG. 29 depicts a Western blot experiment using mAb16-2C9C3 and mAb260-6F9F6 antibodies on cell lysate and recombinant DEK protein. 30 μgof whole cell lysate from 5637 DEK-Sh cell line, 5637 Ns-Sh and 5 μg ofrecombinant DEK protein was run on a gel and analyzed by Western blotusing mAb16-2C9C3 and mAb 260-6F9F6 antibodies. The assay detects bothmonoclonal antibodies against DEK in 5637 NsSh, but does not detect anyof the antibodies against DEK in 5637 DEK-Sh cell lysate, depicting thatmAb16-2C9C3 and mAb 260-6F9F6 are specific for DEK.

FIGS. 30A-30B depict the detection of DEK in DEK spiked urine and DEKspiked PBS using a DEK sandwich ELISA.

FIG. 30A specifically depicts graphical representations of the resultsof 300 μl of various DEK concentrations were spiked into urine and wereanalyzed by DEK sandwich ELISA. rDEK was detected in spiked DEK neaturine with a sensitivity of ˜4 ng/ml.

FIG. 30B specifically depicts graphical representations of the resultsof 300 μl of various DEK concentrations were spiked into PBS wereanalyzed by DEK sandwich ELISA. rDEK was detected in spiked DEK PBS witha sensitivity of ˜2 ng/ml.

FIG. 31 depicts the diagrammatical representation of the DEK sandwichELISA. The capture antibody, mAb16-2C9C3, is immobilized on an ELISAplate followed by the addition of urine containing the DEK protein. Thecaptured DEK antigen is detected by the detection antibody,mAb260-6F9F6, using an HRP linked strepavidin antibody (SA antibody) andTMB as the substrate, the complex is detected by reading absorbance at450 nm.

FIG. 32 depicts the diagrammatical representation of the location ofCDR's (Complementarity Determining Regions) within the variable andconstant regions. The light chain of variable and constant regions isdesignated as V_(L) and C_(L), respectively, and the heavy chain ofvariable and constant regions is designated as V_(H) and C_(H),respectively. In total, there are 6 different CDR sequences present inan antibody.

FIG. 33A depicts the graphical representations of the distributionconcentration of DEK protein in ng/mL in the urine of bladder cancerpatients and control group. 300 μl of neat urine from patients withbladder cancer and control group (healthy individuals and patients withother non-bladder cancer diseases) was analyzed by DEK sandwich ELISA.The absorbance reading was plotted on a standard curve to determine theconcentration of DEK in the urine of tested samples in the two groups(TCC and Control).

FIG. 33B depicts the graphical representations of the distribution ofabsorbance readings of the urine samples in the two groups (TCC andControl) tested by the developed DEK Sandwich ELISA. 300 μl of neaturine from patients with bladder cancer and control group (healthyindividuals and patients with other non bladder cancer diseases) wasanalyzed by DEK sandwich ELISA.

FIG. 33C depicts the receiver operating characteristic (ROC) analysis ofclinical samples comprising of TCC and control group. The ELISA assaydetects the DEK protein in bladder cancer patients with a sensitivity of82.3% and specificity of 70.58%. The assay has a positive predictiverate (PPV) of 75% and a negative predictive value of 84.6%

FIG. 34 depicts a graphical representation of the similarity ofsynthetic urine and healthy urine by using the DEK sandwich ELISA.Recombinant DEK (rDEK) was spiked into synthetic urine and into healthyurine samples and was then analyzed using the DEK sandwich ELISA. rDEKspiked synthetic urine and healthy urine samples had similar assaysensitivity.

FIG. 35A depicts the nucleotide sequence for the heavy chain of mAb16-2C9C3, including CDRs 1-3.

FIG. 35B depicts the amino acid sequence for the heavy chain of mAb16-2C9C3, including CDRs 1-3.

FIG. 36A depicts the nucleotide sequence of the light chain of mAb16-2C9C3, including CDRs 1-3.

FIG. 36B depicts the amino acid sequence for the light chain of mAb16-2C9C3, including CDRs 1-3.

FIG. 37A depicts the nucleotide sequence for the heavy chain of mAb260-6F9F6, including CDRs 1-3.

FIG. 37B depicts the amino acid sequence for the heavy chain of mAb260-6F9F6, including CDRs 1-3.

FIG. 38A depicts the nucleotide sequence of the light chain of mAb260-6F9F6, including CDRs 1-3.

FIG. 38B depicts the amino acid sequence for the light chain of mAb260-6F9F6, including CDRs 1-3.

DETAILED DESCRIPTION OF THE INVENTION

The present invention can be better understood from the followingdescription of preferred embodiments, taken in conjunction with theaccompanying drawings. It should be apparent to those skilled in the artthat the described embodiments of the present invention provided hereinare merely exemplary and illustrative and not limiting.

DEFINITIONS

The following terms shall have the meanings as defined hereunder:

As used herein, the term “DEK” refers to a protein with one SAP domain.DEK protein binds to cruciform and superhelical DNA and induces positivesupercoils into closed circular DNA and involves in splice siteselection during mRNA processing. DEK protein encompasses two isoforms(i.e., DEK isoform 1 and DEK isoform 2) (See, FIG. 4). In humans,isoforms 1 and 2 represent splice variants of DEK that are encoded by aDEK gene (NCBI Accession No. NW_001838973.1) (i.e., the DEK gene islocated on chromosome 6p22). NCBI Accession No. for DEK protein isoform1 is NP_003463.1 (SEQ ID NO: 3). NCBI Accession No. for DEK proteinisoform 2 is NP_001128181.1 (SEQ ID NO: 2). The nucleotide sequence, aswell as the protein sequences, are incorporated by reference herein.

As used herein, the term “precipitation” refers to the condensation of asolid in a solution. Such solid is commonly refers to as “precipitate.”

As used herein, the term “chemical-induced precipitation” refers tousing a chemical compound (e.g., acetone) that causes protein toprecipitate from a solution. The precipitated protein is then collectedby centrifugation (i.e., pellet). The protein pellet may be re-dissolvedin a buffer (i.e., to re-fold protein) to form a solution compatiblewith downstream protein analysis such as Western blot analysis.

As used herein, the term “acetone” refers to the organic compound withthe formula (CH₃)₂CO. Acetone is commonly used in inducing precipitation(i.e., causing protein to precipitate from a solution).

As used herein the term “TCA” refers to trichloroacetic acid that iscommonly used to precipitate proteins in serum.

As used herein, the term “triethanolamine” refers to an organic chemicalcompound which contains a tertiary amine and a triol. A triol is amolecule with three alcohol groups. Like other amines, triethanolamineis a strong base due to the lone pair of electrons on the nitrogen atom.

As used herein, the term “filtration” refers to a mechanical or physicaloperation used for separating solids from fluids by interposing a medium(e.g., filter membrane) through which only the fluid can pass. Oversizesolids in the fluid are retained. Filter membrane may have differentcut-off pore size, for example, 30 kD or 3 kD cut-off.

As used herein, the term “Western blot assay” refers to an analyticaltechnique used to detect specific proteins in a given sample of tissuehomogenate or extract. It utilizes gel electrophoresis to separateeither native proteins or denatured proteins by their lengths or 3-Dstructures. The separated proteins are transferred to a membrane(typically nitrocellulose or PVDF), and are detected using antibodiesspecific against a target protein.

As used herein, the term “antibody” refers to an immunoglobulin producedby B cells and has structural units of two large heavy chains and twosmall light chains. There are two general classes of antibody; namely,monoclonal antibody and polyclonal antibody. Monoclonal antibodies (mAb)refer to monospecific antibodies that are the same because they are madeby identical immune cells that are all clones of a unique parent cell.Monoclonal antibodies are typically made by fusing myeloma cells withthe spleen cells from a mouse that has been immunized with the desiredantigen. Polyclonal antibodies are antibodies obtained from different Bcells. They are a combination of immunoglobulins secreted against aspecific antigen, each identifying a different epitope. Animalsfrequently used for polyclonal antibody production include goats, guineapigs, rabbits, horses, sheep and the like. Rabbit is the most commonlyused laboratory animal for this purpose.

As used herein, the term “protein” refers to a chain of at least twoamino acids. The terms “polypeptide,” “peptide,” or “protein” are usedinterchangeably.

As used herein, the term “bladder cancer” refers to a cancerous tumor inthe bladder. For purposes of this application, bladder cancer is notintended to be limited to cancer of any specific types (i.e., includemany types of cancer in the bladder such as transitional cell carcinoma(TCC), squamous cell carcinoma, adenocarcinoma and combinationsthereof).

As used herein, the term “TCC” refers to transitional cell carcinoma(also known as urothelial cell carcinoma or UCC). It is a type of cancerthat typically occurs in the urinary system: the kidney, urinarybladder, and accessory organs. It is the most common type of bladdercancer and cancer of the ureter, urethra, and urachus. TCC often arisesfrom the transitional epithelium, a tissue lining the inner surface ofthese hollow organs.

As used herein, the term “HxTCC” refers to patients that have apreviously history of TCC.

As used herein, the term “UroTSA” refers to a cell line isolated from aprimary culture of normal human urothelium through immortalization witha construct containing the SV40 large T antigen. It proliferates inserum-containing growth medium as a cell monolayer with little evidenceof uroepithelial differentiation.

As used herein, the term “UroTSA DEK-V5” refers to over-expression ofDEK in UroTSA cells.

As used herein, the term “UroTSA DEKsh” refers to UroTSA cells that havebeen transfected with silencing RNA against native DEK mRNA.

As used herein, the term “CAP” refers to human prostate cancer. The term“HxCAP” refers to patients who had previous history of sufferingprostate cancer.

As used herein, the term “RCC” refers to renal cell carcinoma (alsoknown as hypernephroma). It is a kidney cancer that originates in thelining of the proximal convoluted tubule. RCC is the most common type ofkidney cancer in adults, responsible for approximately 80% of cases. Theterm “HxRCC” refers to patients who had previous history of sufferingrenal cell carcinoma.

As used herein, the term “BPH” refers to benign prostatic hyperplasiaand is synonymous with “benign enlargement of the prostate” (BEP), and“adenofibromyomatous hyperplasia.” All of these diseases are manifestedby an increase in size of the prostate, often in middle-aged and elderlymen.

As used herein, the term “ELISA” (also known as Enzyme-linkedimmunosorbent assay) refers to a biochemical technique used mainly todetect the presence of an antibody or an antigen in a biological sample.For purposes of this application, the ELISA technique is used for thedetection of DEK protein (which is an antigen).

As used herein, the term “indirect ELISA” (also known as Antigen Downmethod) refers to a situation where an unknown amount of antigen isaffixed (i.e., immobilized) to a solid surface, and then a specificantibody (that recognizes the antigen) is added onto the surface so asto allow the forming an antigen-antibody complex. The antigen-antibodycomplex is detected by a secondary antibody. Detection may be achievedby direct linking an enzyme to the secondary antibody or indirect viaanother antibody with an enzyme. The enzyme often converts to somedetectable signal, most commonly a color change in a chemical substrate.

As used herein, the term “sandwich ELISA” (also known as Capture ELISA)refers to immobilizing a capture antibody (specific for the antigen)onto a solid support followed by addition of an amount of antigen. Thebound antigen is then detected by a second antibody (i.e., detectionantibody) which recognizes a region on the antigen that is differentfrom that of the capture antibody. The captured antigen is detected bythe detection antibody which can be covalently linked to an enzyme, orcan itself be detected by addition of a secondary antibody which islinked to an enzyme.

As used herein, the term “anti-DEK antibody” refers to an antibody thatrecognizes DEK protein. There are two DEK isoforms (i.e., DEK isoform 1which contains amino acids 1-375 and DEK isoform 2 which lacks aminoacids 49-82 of the DEK isoform 1). The anti-DEK isoform 1 antibody israised against a peptide corresponding to the amino acids of DEK isoform1 protein. The anti-DEK isoform 2 antibody is raised against a peptidecorresponding to amino acids of the DEK isoform 1 protein, with theexception of the amino acids 49-82.

As used herein, the term “neat urine” refers to urine sample collectedfrom healthy individuals without any dilution or further concentrationof the obtained sample. The developed sandwich ELISA with specificmonoclonal antibodies provides a high sensitivity of <50 ng/mL andpermits the use of neat urine in the ELISA.

As used herein, the term “synthetic urine” refers to a prepared solutionthat mimics human urine. The synthetic urine contains salt (0.9% NaCl)and human albumin (2%) in water, which represent the main constituentsof human urine.

As used herein, the term “clone” refers to a single hybrid cell formedby the fusion of an antibody producing B cells with a myeloma cell andis capable of proliferating indefinitely to produce unlimited quantitiesof identical antibodies.

The present invention is directed to a novel and non-obvious method todetect DEK protein in a urine sample in humans. The present methodcomprises the steps of: (a) forming a precipitate from a urine samplewith a chemical compound selected from the group consisting of acetone,trichloroacetic acid, ethanol, methanol/chloroform and ammonium sulfate;(b) re-suspending said precipitate in a polar solvent to form asolution, said solution has a final volume that is 10-50 fold less thanthat of said urine sample; (c) concentrating said solution 2-10 fold byfiltration; and (d) detecting DEK in said concentrated solution using ananti-DEK antibody in a Western blot assay.

To the best of the present inventors' knowledge, this represents thefirst report for detection of DEK in a human urine sample. Using a cDNAmicroarray system, Sanchez-Carbayo et al. in 2003 reported that DEK gene(among many other genes) is increased in superficial tumors duringprogression of bladder cancer. Although it is logical to deduce that DEKprotein may be presented in urine, no one has successfully documentedsuch a finding and reported the presence of DEK protein in urine.Because DEK is an intracellular protein (i.e., not secreted orreleased), a mere increase in DEK mRNA in bladder cancer may notcorrespondingly produce DEK protein in urine. It is also plausible thatthe dilution of DEK protein in urine exists far below the detectionlimits of any assay. To date, there is no published literaturedescribing DEK protein expression in multiple low and high grade bladdertumors and in the urine of bladder cancer patients. There are no reportsof DEK protein being detected in urine by Western blot assay, ordiagnosing/detecting bladder cancer by detecting DEK protein in urinesamples. The present method therefore has made a significant improvementover the prior art and clearly documented the presence of DEK in urine(which has not previously known to exist).

Our present finding is surprising because the inventors of thisapplication discovered that the sequential order of concentrating aurine sample is critical for the DEK detection. Specifically, no DEKprotein is detectable in urine when a urine sample is concentrated byeither chemical-induced precipitation or filtration-inducedprecipitation alone. DEK protein is also not detectable when a urinesample is first concentrated by filtration followed by achemically-induced precipitation step. The present inventors discoveredthat a urine sample must be concentrated first by (i) chemical-inducedprecipitation, followed by (ii) filtration-induced concentration. Theunderlying mechanism for this observation is unclear.

Using a Western blot assay, the present inventors could not detect DEKprotein in either urine pellets or urine supernatants. DEK protein wasnot detectable even when urines were concentrated by a chemical-inducedprecipitation method. Consecutive precipitations made no difference.These observations suggest that a mere multi-fold concentration of urinedoes not lead to a successful detection of DEK. Similarly, when urineswere concentrated either by single filtration or consecutive filtration,also failed in DEK detection in urine.

Combination of concentrating first by filtration and then bychemical-induced precipitation was found to be ineffective in detectingDEK protein. Only when the urine sample was first concentrated bychemical-induced precipitation followed by filtration-inducedconcentration allows DEK protein detectable by Western blot assay.

The present invention provides a unique sequence of concentrating stepsthat are vital in DEK detection in urine. Without wishing to be bound toa theory, it is believed that a multitude of factors may play a role.These factors include (i) fold concentrations of urine, (ii) DEK proteinconformation (i.e., tertiary protein structure of DEK) and (iii) saltconcentrations. To be detectable, DEK protein may need a sufficient foldconcentration of a urine sample. In chemical-induced precipitation,large amounts of chemical (e.g., acetone) are often needed to achievethe necessary fold concentration. Given this constraint, a singlechemical-induced precipitation may not reach the necessary foldconcentration. Filtration-induced concentration may cure thisdeficiency. In the process of chemical-induced precipitation, there maybe a slight distort in tertiary protein structure and thus affectWestern blot analysis. Filtration-induced concentration, althoughmaintain the optimal protein tertiary structure, suffers fromcontamination with high salts. Our data with conductivity supports thiscontention. High salt concentration may affect Western blot assay in DEKdetection.

The present inventors also surprisingly discovered a correlation betweenthe presence of DEK protein in urine and detection/diagnosis of bladdercancer in humans. Specifically, the present invention provides a methodof detecting bladder cancer in a human, comprising the steps of: (a)obtaining a urine sample from a human; (b) forming a precipitate fromsaid urine sample with a chemical compound selected from the groupconsisting of acetone, trichloroacetic acid, ethanol,methanol/chloroform and ammonium sulfate; (c) re-suspending saidprecipitate in a polar solvent to form a solution, said solution has afinal volume that is 10-50 fold less than that of urine sample; (d)concentrating said solution 2-10 fold by filtration; and (e) detectingDEK in said concentrated solution using an anti-DEK antibody in aWestern blot assay, wherein the presence of DEK protein in said urinesample is an indicative of a bladder cancer in said human.

The present non-invasive method for detecting DEK in urine has anexceedingly high sensitivity (i.e., 79%) and specificity (i.e., 83%) ascompared to other urine-based assays. Currently, there are five (5)commercial tests that detect biomarkers of bladder cancer in urine.These include: (i) NMP22 ELISA (detects nuclear mitotic apparatusprotein) has 47-100% sensitivity and 60-80% specificity; (ii)BladderChek® dipstick test (detects nuclear mitotic apparatus protein)has 49.5% sensitivity and 87.3% specificity; (iii) BTA-Stat® test(detects complement factor H-related protein) has 50-70% specificity;(iv) urinary bladder cancer test (detects cytokine 8 and cytokine 18 byELISA) has 56% sensitivity and 97% specificity; and (v) UroVysion® (afluorescence in situ hybridization (FISH) based assay that detectsamplification of chromosomes 3, 7 and 17 and loss of chromosome region9p21) has 68-81% sensitivity and 79-96% specificity.

Our ELISA study reveals clearly that DEK isoform 2 protein is present inurine of humans who suffers from bladder cancer disorder. The finding issurprising and unexpected because while both isoform 1 and isoform 2 arepresent in tumor tissues, only isoform 2 is present in urine. Theunderlying mechanistic basis for our finding is unclear. It is unlikelythat only the DEK isoform 2 is secreted or released during thepathogenesis of bladder cancer. Nevertheless, this constitutes the firstreport that DEK isoform 2 in urine bears a high correlation with humanbladder cancer disorders.

Our ELISA is sensitive to detect DEK isoform 2 protein when the proteinis in its proper tertiary conformation state. While ELISA is capable ofdetecting recombinant DEK protein spiked into a human urine, anyacetone-treatment (i.e., during the concentrating step) would abolishthe ability of ELISA in detecting DEK protein. This suggests that whileour Western blot assay may tolerate some degree of tertiary proteinconformation alternation (e.g., under denaturing conditions andacetone-treatment), ELISA assay can only detect DEK protein withfiltration-induced concentration.

Urine may be conveniently collected from a human subject using asuitable container with a sufficient volume capacity for DEK proteinassay. Commercially available urine containers may be used. In oneembodiment, a urine container may contain a cap to prevent spilling anda means to allow the collected urine sample to be transported. Urine maybe stored under appropriate conditions. In one embodiment, the containermay be capable of withstanding freezing conditions (e.g., −80° C.). Forpurposes of the present assay, a sufficient urine volume may range from15 ml to 75 mL. In one preferred embodiment, urine volume of between 20mL to 40 mL is adequate.

Time of urine collection is not critical. In one embodiment, urine iscollected as first void urine (i.e., in the morning). First void urineis believed to contain a greater amount of proteins, and may thereforeincrease the ability of detection for urine-based biomarkers. In anotherembodiment, urine may be collected during daytime or before bedtime.

Freshly collected urine (i.e., urine samples immediately aftercollection) may be used. Alternatively, frozen urine may be used (i.e.,after thawing of frozen urine samples). For purposes of thisapplication, we detect no difference between freshly collected urine andthawed urine. For convenient purposes, collected urine is stored between−20° C. and −80° C. Urine may be conveniently stored for at least6-month duration.

In one embodiment, a protease inhibitor may be added to a urine sampleand in an amount sufficient to prevent potential protein degradation ofurine proteins. Suitable protease inhibitor includes, but not limitedto, aprotinin, pepstatin, phenylmethanesulfonyl fluoride, chymostatin,and the like. In an alternative embodiment, a cocktail of suitableprotease inhibitors may be used. For example, commercially availableprotease inhibitor cocktail (“Complete Protease Inhibitor CocktailTablets”) (Roche; cat. no. 11836153001) may be used. In one embodiment,protease inhibitors may be added immediately after urine is collected.In yet an embodiment, protease inhibitors may be added after urine isthawed.

One skilled in the art would know how to optimize the suitable amount ofprotease inhibitors needed to prevent potential protein degradation inurine. In one embodiment, protease inhibitors are added to achieve afinal concentration of 10 μg/ml to 5 mg/ml. In a preferred embodiment,protease inhibitors may be added to achieve a final concentration of 250μg/ml to 750 μg/ml. In another preferred embodiment, protease inhibitormay be added to achieve a final concentration of 1 mg/ml.

Urine may be turbid which may be a symptom of a bacterial infection. Aturbid urine may be caused by crystallization of salts such as calciumphosphate. In one embodiment, potential crude debris present in a turbidurine sample may be cleared prior to the concentrating steps. In oneembodiment, collected urine samples may simply be passed through acloth, paper, tissue and the like. For example, urine may be cleared bypassing through a Kimwipe® (Kimberly-Clarke, Dallas, Tex.) prior to theurine concentrating steps.

One aspect of the present invention provides a step of concentratingurine using a chemical compound. Urine may be concentrated by achemical-induced precipitation. Chemical-induced precipitation generallyinvolves adding a chemical compound to a urine sample to cause urineproteins to form a precipitate. Urine precipitates are visible with anaked eye. Suitable chemical compounds include, without limitation,acetone, trichloroacetic acid, ethanol, methanol chloroform, ammoniumsulfate and the like. In a preferred embodiment, the chemical compoundis acetone or trichloroacetic acid. Methanol/chloroform is a solventmixture, preferably comprising methanol and chloroform in avolume-to-volume ratio of 2:3. Other optimal volume-to-volume ratio ofmethanol and chloroform may be determined by one of ordinary skill inthe art in so far as they function to induce urine protein toprecipitate,

To aid in urine concentration, chemical compound is used at an amountsufficient to induce the formation of a precipitate. In one embodiment,chemical compound is added to a urine sample to achieve a ratio ofchemical compound volume to urine sample volume (vol/vol ratio) ofbetween 10:1 to 2:1. In another embodiment, chemical compound is used ata vol/vol ratio of between 5:1 to 2:1. In a preferred embodiment, thevol/vol ratio is 2:1 (e.g., 50 ml acetone is added to 25 ml urine).

To enhance precipitation formation, it is found that adding ice-coldchemical compound is preferred. In one preferred embodiment, ice-coldacetone is used as the precipitating chemical. In another embodiment,ice-cold acetone is added to the urine sample and the resulting solutioncontinued to be chilled at between −20° C. and −80° C. In yet anotherembodiment, ice-cold acetone is added to the urine sample and thesolution chilled at −40° C.

In one embodiment, the resulting chemical-urine solution (e.g.,acetone-urine) is chilled for an additional of 0.5-4 hours to cause theprecipitates to be formed. In a preferred embodiment, the solution ischilled for 1-3 hours. In another preferred embodiment, the solution ischilled for 1.5-2 hours.

Methods are known in the art to collect precipitates as pellets afterthe step of chemical-induced precipitation. For example, a briefcentrifugation (e.g., 12,000 rpm, 15 minutes) may be used to collect theprecipitated proteins.

Another aspect of the present invention provides a step of furtherconcentrating urine using a filtration method. Prior to thefiltration-induced concentration, the pelleted proteins may convenientlybe re-suspended in a suitable re-suspension buffer. Without wishing tobe bound by a theory, the re-suspension buffer is believed to enhancerefolding of the precipitated proteins. Re-suspension may help torestore and reform the precipitated proteins into a proper tertiaryprotein structure.

Ideally, the re-suspension buffer may match the pH of urine. The pH ofurine is close to neutral (pH 7) but can normally vary between 4.4 and8. A diet high in citrus, vegetables, or dairy can increase urine pH.Some drugs can increase urine pH, including acetazolamide, potassiumcitrate, and sodium bicarbonate. On the other hand, a diet high in meator cranberries can decrease urine pH. Drugs that can decrease urine pHinclude ammonium chloride, chlorothiazide diuretics, and methenaminemandelate. In one embodiment, the re-suspension buffer has a pH ofbetween 5-9. More preferably, the re-suspension buffer has a pH ofbetween 6-8. More preferably, the re-suspension buffer has a pH of 7.5.

One skilled in the art would recognize the use of common buffers tomaintain pH of the re-suspension buffer. A buffer solution is an aqueoussolution consisting of a mixture of a weak acid and its conjugate baseor a weak base and its conjugate acid. Buffer solutions are used as ameans of keeping pH at a nearly constant value in a wide variety ofchemical applications. Exemplary common buffer includes triethanolamine,TRIS, HEPES, MOPS, and the like. Preferably, the re-suspension buffer isisotonic.

In one embodiment, the re-suspension buffer is an organic chemicalcompound which is both a tertiary amine and a triol such astriethanolamine. Preferably, the re-suspension buffer contains 10 mMtriethanolamine. In another embodiment, the re-suspension buffer mayinclude a sugar to enhance tonicity. Exemplary sugar includes, but notlimited to, sucrose. Preferably, the re-suspension buffer contains 250mM sucrose. An exemplary re-suspension buffer is a solution of 10 mMtri-ethanolamine and 250 mM sucrose.

The pelleted proteins are re-suspended at a minimal volume that is muchless than the original urine sample volume. One skilled in the art wouldrecognize a minimum optimal volume in re-suspending the pellet proteins.In one embodiment, the pelleted protein is re-suspended in a volume ofre-suspension buffer of 500 μl. The volume of the re-suspension buffermay range from 5-50 fold less than that of the original urine sample.Preferably, the volume of the re-suspension buffer ranges from 20-40fold less than the original urine sample volume. More preferably, thevolume of the re-suspended is 30 fold less than the original urinesample volume (e.g., pelleted proteins from 60 mL urine is re-suspendedin 500 μl re-suspension buffer).

Re-suspended concentrated urine is further concentrated. In one aspect,the present invention provides a step of concentrating urine byfiltration. Filtration-induced concentration may be accomplished throughthe use of spin filter concentration units. Examples of commerciallyavailable spin-filter concentration units include units sold under thetradenames Microcon®, Centricon® and Centriprep®.

In one embodiment, the spin filter has a molecular weight cut-off ofbetween 1 kD and 40 kD. In another embodiment, the spin filter has amolecular weight cut-off of 3 kD. In another embodiment, the spin filterhas a molecular weight cut-off of 30 kD.

Ideally, filtration-induced concentration is used to achieve an increaseof concentration between 2-fold to 10-fold. In one embodiment,concentration is increased between 4-fold to 8-fold. In a preferredembodiment, concentration is increased 5-fold.

Protein concentrations of urine samples may be conveniently quantifiedby methods that are known to the art including assays that arecommercially available. For example, one commercially available kit isthe BCA assay kit (Pierce, Thermo Fisher Scientific, Rockford, Ill.).

In one aspect, the present invention provides an assay to detect DEKprotein present in the concentrated urine samples. In one embodiment,protein present in the filtration-concentrated sample is adjusted to alevel of 10-1,000 μg/ml. Preferably, the protein concentration in thefiltration-concentrated sample is 100 μg/ml.

DEK protein may be detected using standard protein detection assays thatare known in the art. These assays include, but not limited to, Westernblot analysis, ELISA, radioimmunoassay, dot-blot assay, and the like.Preferably, DEK protein is detected by Western blot analysis.

After urine samples are treated (i.e., subject to chemical-inducedprecipitation and filtration-induced concentration), the proteins areseparated using SDS-PAGE gel electrophoresis. The technology of SDS-PAGEgel electrophoresis is well known in the art. Approximately 5 μg to 100μg of total protein is run on a SDS-PAGE gel. Preferably, 10 μg to 75 μgof total protein is used. More preferably, 25 μg of total protein isused. The conditions for SDS-PAGE gel electrophoresis can beconveniently optimized by one skilled in the art. In one embodiment,SDS-PAGE gel is run at 100 V for 90 min of 400 mA for 90 minutes.Optimally, gel electrophoresis may be performed under denaturingconditions. SDS-PAGE gel electrophoresis conditions are well known bythose skilled in the art and can be conveniently optimized.

Following gel electrophoresis, the proteins present in the gels may betransferred onto a suitable solid surface such as nitrocellulose paper,nylon membrane, PVDF membrane and the like. Preferably, PVDF membrane isused. The conditions for protein transfer after SDS-PAGE gelelectrophoresis may be optimized by one skilled in the art.

Western blot may be used to detect DEK protein in the concentrated urine(after SDS-PAGE). A first antibody specific for the protein of interest(e.g., DEK) is employed. The first antibody may be either a monoclonalantibody or polyclonal antibody. Antibodies against the proteinbiomarker can be prepared using standard protocols or obtained fromcommercial sources. Techniques for preparing mouse monoclonal antibodiesor goat or rabbit polyclonal antibodies (or fragments thereof) are wellknown in the art.

Membrane may be incubated with a blocking solution before the incubationwith the first antibody. Blocking solution may include agents thatreduce non-specific binding of antibody. For example, blocking solutionmay include 5% skim milk in PBST (0.1% Tween-20).

Bound proteins (e.g., 10-100 μg) on the membrane are incubated with afirst antibody in a solution. In one embodiment, the first antibody isused at a concentration of 0.2-2 μg/mL. Preferably, the first antibodyis used at a concentration of 1 μg/mL.

Incubation conditions may be optimized to maximize the binding of thefirst antibody with the bound biomarker proteins. In one embodiment, theincubation time is 1-6 hours. In a preferred embodiment, the incubationtime is 2 hours.

After incubation with the first antibody, unbound antibody may beconveniently removed by washing. In one embodiment, the washing solutionmay include PBST.

Protein biomarker-first antibody complex (e.g., DEK-anti-DEK antibody)may be detected by incubation with a second antibody that is specificfor the first antibody. The second antibody may be a monoclonal antibodyor a polyclonal antibody (e.g., mouse, rabbit, or goat). In oneembodiment, the second antibody may carry a label which may be adirectly detectable label or may be a component of a signal-generatingsystem. In another embodiment, the second antibody is a goat anti-rabbitantibody or goat anti-mouse antibody that is labeled with a peroxidase.Such labeled antibodies and systems are well known in the art.

Direct detectable label or signal-generating systems are well known inthe field of immunoassay. Labeling of a second antibody with adetectable label or a component of a signal-generating system may becarried out by techniques well known in the art. Examples of directlabels include radioactive labels, enzymes, fluorescent andchemiluminescent substances. Radioactive labels include ¹²⁴I, ¹²⁵I,¹²⁸I, ¹³¹I, and the like. A fluorescent label includes fluorescein,rhodamine, rhodamine derivatives, and the like. Chemiluminescentsubstances include ECL chemiluminescent.

In another aspect, the present invention provides a method of detectingand diagnosing bladder cancer. This is accomplished by obtaining andtesting a urine sample and detecting the presence of DEK protein in theurine sample by the detection method provided herein. The presence ofDEK protein in the urine sample indicates that the patient tested issuffering from bladder cancer. Thus, the present invention provides anefficient, non-invasive method for the detection and diagnosis ofbladder cancer by detecting DEK protein in a urine sample.

Our Western blot assay results suggest that urine contains only DEKisoform 2 in individuals suffering from bladder cancer. In our assay, weused a monoclonal anti-DEK antibody that specifically recognize DEKisoform 1 (but not DEK isoform 2) (i.e., the antibody was raisedspecifically to a region that is present only in DEK isoform 1 but notDEK isoform 2). The polyclonal anti-DEK antibody that was usedrecognizes both DEK isoforms. Because both monoclonal and polyclonalanti-DEK antibodies recognized DEK proteins in both bladder cancer cellculture and bladder cancer tissue samples, this implies that DEK isoform1 and DEK isoform 2 are present in these cells and tissues.

However, only the polyclonal anti-DEK antibody (but not the monoclonalanti-DEK antibody) recognized DEK protein in urine samples from patientssuffering bladder cancer, this suggests urine contains DEK isoform 2,but not DEK isoform 1.

ELISA Assay

Detection of DEK protein in urine may be accomplished by techniquesknown in the art, e.g., ELISA (enzyme-linked immunosorbent assay),Western blots, and the like.

As appreciated by one skilled in the art, an enzyme-linked immunosorbentassay (ELISA) may be employed to detect proteins in urine, specificallyDEK protein in urine. In one aspect, the present invention provides anELISA in detecting DEK protein (i.e., DEK isoform 2 protein) in humanurine. The presence of DEK isoform 2 protein is an indication of bladdercancer.

In one embodiment, the present invention provides an initial step of anELISA in which an anti-DEK antibody is immobilized onto a surface (forexample by passive adsorption known as coating). For purposes of thisapplication, exemplary DEK is isoform 2 and the fragment thereof.Anti-DEK antibody may recognize recombinant full-length DEK protein aswell as fragments thereof via its recognition of specific epitopes.Immobilization of anti-DEK antibody may be performed on any inertsupport (or solid support) that is useful in immunological assays.Examples of commonly used inert supports include small sheets, Sephadexand assay plates manufactured from polyethylene, polypropylene orpolystyrene. In a preferred embodiment the immobilized anti-DEK antibodyis coated on a microtiter plate that allows analysis of several samplesat one time. More preferably, the microtiter plate is a microtest96-well ELISA plate, such as those sold under the name Nunc Maxisorb orImmulon.

Antibody immobilization is often conducted in the presence of a bufferat an optimum time and temperature optimized by one skilled in the art.Suitable buffers should enhance immobilization without affecting theantigen binding properties. Sodium carbonate buffer (e.g., 50 mM, pH9.6) is a representative suitable buffer, but others such as Tris-HClbuffer (20 mM, pH 8.5), phosphate-buffered saline (PBS) (10 mM, pH7.2-7.4) are also used. Optimal coating buffer pH will be dependent onthe antigen(s) being immobilized. Optimal results may be obtained when abuffer with pH value 1-2 units higher than the isoelectric point (pI)value of the protein is used. Incubation time ranges from 2-8 hours toovernight. Incubation may be performed at temperatures ranging from4-37° C. Preferably, immobilization takes place overnight at 4° C. Theplates may be stacked and coated long in advance of the assay itself,and then the assay can be carried out simultaneously on several samplesin a manual, semi-automatic, or automatic fashion, such as by usingrobotics.

Blocking agents are used to eliminate non-specific binding sites inorder to prevent unwanted non-specific antibody binding to the plate.Examples of appropriate blocking agents include detergents (for example,Tween-20, Tween-80, Triton-X 100, sodium dodecyl sulfate), gelatin,bovine serum albumin (BSA), egg albumin, casein, non-fat dried milk andthe like. Preferably, the blocking agent is BSA. Concentrations ofblocking agent may easily be optimized (e.g. BSA at 1-5%). The blockingtreatment typically takes place under conditions of ambient temperaturesfor about 1-4 hours, preferably 1.5 to 3 hours.

After coating and blocking, urine from control subjects or patientssuspected of bladder cancer are added to the immobilized antigens in theplate. Concentrated urine suspended in Phosphate Buffered Saline (PBS)containing 0.5% BSA, 0.05% TWEEN 20® detergent may be used. TWEEN 20®acts as a detergent to reduce non-specific binding.

The conditions for incubation of the biological sample and immobilizedantigen are selected to maximize sensitivity of the assay and tominimize dissociation. Preferably, the incubation is accomplished at aconstant temperature, ranging from about 0° C. to about 40° C.,preferably from about 22 to 25° C. to obtain a less variable, lowercoefficient of variant (CV) than at, for example, room temperature. Thetime for incubation depends primarily on the temperature, beinggenerally no greater than about 10 hours to avoid an insensitive assay.Preferably, the incubation time is from about 0.5 to 3 hours, and morepreferably 1.5-3 hours at room temperature to maximize binding toimmobilized capture antigen.

Following incubation of the biological sample (urine) and immobilizedanti-DEK antibody, unbound biological sample is separated from theimmobilized antibody by washing. The solution used for washing isgenerally a buffer (“washing buffer”) with a pH determined using theconsiderations and buffers described above for the incubation step, witha preferable pH range of about 6-9. Preferably, pH is 7. The washing maybe done three or more times. The temperature of washing is generallyfrom refrigerator to moderate temperatures, with a constant temperaturemaintained during the assay period, typically from about 0-40° C., morepreferably about 4-30° C. For example, the wash buffer can be placed inice at 4° C. in a reservoir before the washing, and a plate washer canbe utilized for this step.

Next, the immobilized capture anti-DEK antibody and biological sample(i.e., urine) are contacted with a detectable antibody at a time andtemperature optimized by one skilled in the art. Detectable antibody mayinclude a monoclonal antibody or a polyclonal antibody. These antibodiesmay be directly or indirectly conjugated to a label. Suitable labelsinclude moieties that may be detected directly, such as fluorochrome,radioactive labels, and enzymes, that must be reacted or derivatized tobe detected. Examples of such labels include the radioisotopes ³²P, ¹⁴C,¹²⁵I, ³H, and ¹³¹I, fluorophores such as rare earth chelates orfluorescein and its derivatives, rhodamine and its derivatives,horseradish peroxidase (HRP), alkaline phosphatase, and the like.Preferably, the detection antibody is a goat anti-human IgG polyclonalantibody that binds to human IgG and is directly conjugated to HRP.Incubation time ranges from 30 minutes to overnight, preferably about 60minutes. Incubation temperature ranges from about 20-40° C., preferablyabout 22-25° C., with the temperature and time for contacting the twobeing dependent on the detection means employed.

The conjugation of such labels to the antibody, including the enzymes,is a standard manipulative procedure for one of ordinary skill inimmunoassay techniques. See, for example, O'Sullivan et al. “Methods forthe Preparation of Enzyme-antibody Conjugates for Use in EnzymeImmunoassay,” in Methods in Enzymology, ed. J. J. Langone and H. VanVunakis, Vol. 73 (Academic Press, New York, N.Y., 1981), pp. 147-166.

In one embodiment, after the complex formation between DEK protein andanti-DEK antibody, the antibody binding to antigen (i.e., DEK protein)is assessed by detecting a label on the primary antibody. In anotherembodiment, the primary antibody is assessed by detecting binding of asecondary antibody or reagent to the primary antibody. In a furtherembodiment, the secondary antibody is labeled. Many means are known inthe art for detecting binding in an immunoassay and are within the scopeof the present invention. For example, to select specific epitopes ofrecombinant or synthetic polypeptide, one may assay antibody binding inan ELISA assay wherein the polypeptides or its fragments containing suchepitope.

In another aspect, the present invention provides direct immobilizingurine samples (containing DEK protein) onto a solid support (e.g.,microtiter plates). This is also known as antigen-down ELISA (i.e.,indirect ELISA). In such an assay, a microtiter plate is coated with asample containing a certain antigen (e.g., DEK protein). After allowingfor adsorption of the antigen onto the plate, and washing off allnon-bound materials, an antibody is added to the plate and the excesswashed off. Prior to addition of the antibody, one skilled in the artwould appreciate blocking non-specific bindings with appropriateblocking agents (e.g., Tween-20, Tween-80, Triton-X 100, sodium dodecylsulfate), gelatin, bovine serum albumin (BSA), egg albumin, casein,non-fat dried milk and the like). The added antibody may have alreadybeen labeled with a reporter molecule to permit the generation of asignal to be read by known techniques (e.g., microtiter plate reader).

An anti-DEK protein is directly added to allow the formation of DEK andanti-DEK antibody complex. Optimal conditions for antigen-antibodycomplex formation may be conveniently adjusted by one of ordinaryskilled in the art. Unbound antibody is removed by washing; generally bya buffer. Detectable antibody may include a monoclonal antibody or apolyclonal antibody. These antibodies may be directly or indirectlyconjugated to a label (as described above).

Alternatively an enzyme conjugated secondary antibody can be used fordetection of antigen-antibody complex. Upon addition of a suitablechromogen substrate, a color develops which is used as an indicator ofthe amount of antigen present.

mAb 260-6F9F6 and mAb 16-2C9C3

In one embodiment, the present invention provides a total of eight (8)anti-DEK monoclonal antibodies, which antibodies recognize and bind toDEK protein. They include mAb 16-1D4F8, mAb 16-1D4F10, mAb 16-DC9C3, mAb260-6C5G8, mAb 260-6D11F2, mAb 260-6F9F6, mAb 320-2B9A8, and mAb320-3E9E11. Using these mAbs in different permutations (either as acapture antibody or detection), we discovered that two (2) of these mAbsare useful in establishing a highly sensitive ELISA (<50 ng/mL). In apreferred embodiment, the present invention provides an ELISA employingmAb 16-2C9C3 as the capture antibody and mAb 260-F9F6 as the detectionantibody.

A natural antibody molecule contains two identical pairs of polypeptidechains, each pair having one light chain and one heavy chain. Each lightchain and heavy chain in turn consists of two regions: a variable (“V”)region involved in binding the target antigen, and a constant (“C”)region that interacts with other components of the immune system. Thelight and heavy chain variable regions come together in 3-dimensionalspace to form a variable region that binds the antigen (for example, areceptor on the surface of a cell). Within each light or heavy chainvariable region, there are three short segments (averaging 10 aminoacids in length) called the complementarity determining regions(“CDRs”). The six CDRs in an antibody variable domain (three from thelight chain and three from the heavy chain) fold up together in 3-Dspace to form the actual antibody binding site which locks onto thetarget antigen. The position and length of the CDRs have been preciselydefined by Kabat, E. et al., Sequences of Proteins of ImmunologicalInterest, U.S. Department of Health and Human Services, 1983, 1987. Thepart of a variable region not contained in the CDRs is called theframework, which forms the environment for the CDRs.

The mAb 16-2C9C3 was further characterized by sequencing its nucleotidesand amino acids. In one embodiment, the present invention provides anisolated anti-DEK antibody (mAb 16-2C9C3) comprising: (i) CDR1 of theheavy chain variable region which has the amino acid sequence of SEQ IDNO:15; (ii) CDR2 of the heavy chain variable region which has the aminoacid sequence of SEQ ID NO:16; (iii) CDR3 of the heavy chain variableregion which has the amino acid sequence of SEQ ID NO:17. In anotherembodiment, the present invention provides an isolated anti-DEK antibody(mAb 16-2C9C3) comprising: (i) CDR1 of the light chain variable regionwhich has an amino acid sequence of SEQ ID NO: 23; (ii) CDR2 of thelight chain variable region which has the amino acid sequence of SEQ IDNO:24; and (iii) CDR3 of the light chain variable region which has theamino acid sequence of SEQ ID NO:25.

In yet another embodiment, the present invention provides an isolatedmonoclonal antibody (mAb 16-2C9C3), comprising: (i) CDR1 of the heavychain variable region which has the amino acid sequence of SEQ ID NO:15;(ii) CDR2 of the heavy chain variable region which has the amino acidsequence of SEQ ID NO:16; (iii) CDR3 of the heavy chain variable regionwhich has the amino acid sequence of SEQ ID NO:17, (iv) CDR1 of thelight chain variable region which has an amino acid sequence of SEQ IDNO: 23; (v) CDR2 of the light chain variable region which has the aminoacid sequence of SEQ ID NO:24; and (vi) CDR3 of the light chain variableregion which has the amino acid sequence of SEQ ID NO:25.

The mAb 260-F9F6 was also further characterized by sequencing itsnucleotides and amino acids. In one embodiment, the present inventionprovides an isolated anti-DEK antibody (mAb 260-F9F6) comprising: (i)CDR1 of the heavy chain variable region which has the amino acidsequence of SEQ ID NO:31; (ii) CDR2 of the heavy chain variable regionwhich has the amino acid sequence of SEQ ID NO:32; (iii) CDR3 of theheavy chain variable region which has the amino acid sequence of SEQ IDNO:33. In another embodiment, the present invention provides an isolatedanti-DEK antibody (mAb 260-F9F6) comprising: (i) CDR1 of the light chainvariable region which has an amino acid sequence of SEQ ID NO: 39; (ii)CDR2 of the light chain variable region which has the amino acidsequence of SEQ ID NO:40; and (iii) CDR3 of the light chain variableregion which has the amino acid sequence of SEQ ID NO:41.

In yet another embodiment, the present invention provides an isolatedmonoclonal antibody (mAb 260-F9F6), comprising: (i) CDR1 of the heavychain variable region which has the amino acid sequence of SEQ ID NO:31;(ii) CDR2 of the heavy chain variable region which has the amino acidsequence of SEQ ID NO:32; (iii) CDR3 of the heavy chain variable regionwhich has the amino acid sequence of SEQ ID NO:33, (iv) CDR1 of thelight chain variable region which has an amino acid sequence of SEQ IDNO: 39; (v) CDR2 of the light chain variable region which has the aminoacid sequence of SEQ ID NO:40; and (vi) CDR3 of the light chain variableregion which has the amino acid sequence of SEQ ID NO:41.

The use of mAb 16-2C9C3 (as a capture antibody) and mAb 260-F9F6 (as adetection antibody) surprisingly yields high sensitivity in DEKdetection. The ELISA affords at least <50 ng/mL limit of detection.Preferably, the ELISA offers 3.9 ng/mL limit of detection. To oursurprise and for reasons unknown, the use of mAb 16-2C9C3 (captureantibody) and mAb 260-F9F6 (detection antibody) does not require aconcentration step of urine. There is no need to concentrate urine(either by filtration or chemical-induced method as described above)prior to the ELISA detection. Neat urine can be used in the ELISA todetect DEK.

Kits

Another aspect of the invention is to provide a kit that may be used todetect DEK protein in urine. The kit according to the present inventionincludes a set of antibodies (i.e., a first antibody and a secondantibody) that are specific for DEK protein. In one embodiment, the kitcontains reagents (e.g., precipitating chemicals such as acetone or TCA)for treating the urine sample so as to enable DEK protein to be detectedfrom the sample. In another embodiment, the kit contains ELISA platesnecessary to perform direct or indirect ELISA to detect DEK protein.

Kits provided herein include instructions, such as a package inserthaving instructions thereon, for using the reagents to prepare and stepsin concentrating a urine sample. Such instructions may be for using thereagents to prepare the urine sample to specifically allow detection ofDEK protein from the urine. In another embodiment, the instructions aredirected to the use of antibodies (either monoclonal or polyclonal) thatrecognize and bind to DEK protein in Western blot analysis or ELISA.

In one embodiment, the present invention provides a kit for detectingbladder cancer in a human, employing mAb 16-2C9C3 as a capture antibodyand/or mAb 260-6F9F6 as a detection antibody for DEK. The providedinstruction guides one skilled artisan to use the mAb 16-2C9C3 and mAb260-6F9F6.

The following examples are provided to further illustrate variouspreferred embodiments and techniques of the invention. It should beunderstood, however, that these examples do not limit the scope of theinvention described in the claims. Many variations and modifications areintended to be encompassed within the spirit and scope of the invention.

EXPERIMENTAL STUDIES Example 1 Western Blot Detection of DEK Protein inBladder Cell Lines

In this series of studies, we optimized the detection of DEK protein ina biological sample (e.g., urine) using Western blot assay. We testedcell lysate extracts obtained from four (4) bladder cancer cell lines(i.e., RT-4, 5637, T-24 and TCCSUP) and examined their DEK proteinexpression. These cells were chosen to represent different stages ofbladder cancer. In addition, we tested cell extracts from bladderepithelial cells transformed with SV-40 T-antigen (i.e., UroTSA),progenitor human epithelial cells (i.e., HBEP cells), and differentiatedHBEP cells for their DEK protein expression. Note that HBEP cells weretreated with 1 mM calcium chloride to prevent cycling in growth media.

Cell extracts from 1×10⁷ cells were obtained using RIPA buffer (i.e., 25mM Tris-HCl (pH 7.6), 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, and0.1% SDS). Protein was quantified using a BCA assay kit (Pierce, ThermoFisher Scientific, Rockford, Ill.). Cell lysates extracts were furtherconcentrated to a concentration of between 4 and 8 μg/μl. 30 μg of thecell extracts were used in the Western blot analysis using an anti-DEKantibody (e.g., an anti-DEK monoclonal antibody; cat #610948) (BDBioscience, San Jose, Calif.).

FIG. 1 shows a Western blot demonstrating the presence of DEK proteinexpressed in four (4) bladder cancer cell lines (i.e., RT-4, 5637, T-24and TCCSUP) as well as the UroTSA and undifferentiated HBEP cells. Inthis Western blot analysis, we used a monoclonal anti-DEK antibody (cat.no. 610948) (BD Bioscience, San Jose, Calif.). Note that DEK protein hasa molecular size of ˜43 kD. DEK protein was not detectable indifferentiated (i.e., non-cancer) HBEP cells. β-actin served as aloading control. In another Western blot analysis, we used a polyclonalanti-DEK antibody (cat. no. A-301-335A) (Bethyl Labs, Montgomery, Tex.).Similar to that in monoclonal antibody study, we observed a similarprofile in DEK protein expression in these cells (data not shown).

Thus, we have developed a Western blot assay that is sensitive andspecific in detecting DEK protein expression using cell lysates extractsobtained from bladder cancer cell lines, using either a monoclonalanti-DEK antibody or a polyclonal anti-DEK antibody.

Example 2 Western Blot Fails to Detect DEK Protein in Cultured Media

In this study, we examined if DEK protein can be released from bladdercancer cells (i.e., secreted from cells). To do so, we first collectedcultured media from two (2) bladder cancer cell lines (i.e., T-24 and5637) and then examined DEK protein expression in these cultured media.One (1) ml of cultured media was collected and briefly centrifuged(3,000 rpm, 5 min) to remove any cellular debris. The cultured media wassubsequently concentrated to 50-fold (i.e., from 500 μl to 10 μl) usinga Microcon® 3K filter (Millipore, Billerica, Mass.). The entire 10 μl ofthe concentrated cultured media sample was loaded in a Western blotassay. DEK protein was examined using a polyclonal anti-DEK antibody(cat. no. A-301-335A) (Bethyl Labs., Montgomery, Tex.). UroTSA (10 μg)whole cell lysate was used as a control.

FIG. 2 shows that DEK protein was not detectable in the cultured mediaof the two (2) bladder cancer cell lines (i.e., T-24 and 5637). DEKprotein was not detectable in the cultured media from the transformedbladder epithelial cells (i.e., UroTSA) and from bladder epithelialcells that were over-expressing DEK protein (UroTSA DEK-V5). This datasuggest that DEK protein is neither secreted nor released from bladdercancer cells. As a negative control, we transfected DEK shRNA (i.e.,small hairpin RNA against DEK) in UroTSA cells in order to shut down DEKprotein expression. No detectable DEK protein was found in the culturedmedia from UroTSA DEK shRNA, confirming that DEK protein may not bereleased from bladder cancer cells.

Example 3 Western Blot Detection of DEK Protein in Bladder Tissues

In this study, we examined if our Western blot assay (see Example 1)could detect DEK protein in bladder tissues obtained from human subjects(e.g., bladder cancer patients or healthy individuals).

Twenty-seven (27) bladder tumor tissue samples were obtained frompatients who suffered from low and high grade transitional cellcarcinoma (TCC). For comparison, twenty-seven (27) normal bladder tissuesamples were obtained from the adjacent sites of the same individuals.Tissue lysate extracts were prepared using RIPA buffer (as describedabove) and the tissue extracts were prepared to a protein concentrationof 2-10 μg/μL. Protein concentration of the tissue lysates extracts wasdetermined using a BCA assay kit (Pierce, Thermo Fisher Scientific,Rockford, Ill.). ˜50 μg of the tissue lysates extracts from each samplewas analyzed for their DEK protein expression in our Western blot assay,using a monoclonal anti-DEK antibody (i.e., cat. no. 610948). In someWestern blot analysis, we used a polyclonal anti-DEK antibody (cat. no.A-301-335A) (Bethyl Labs, Montgomery, Tex.) and observed the same DEKprotein expression. 10 μg of the UroTSA cell lysates was used as acontrol.

FIG. 3 shows the Western blot of the DEK protein in ten (10)representative bladder tumor tissues using the monoclonal anti-DEKantibody. In total, we have examined twenty-seven (27) bladder tissuesamples. Out of these bladder tissue samples, DEK protein expression wasdetected in twenty-two (22) bladder tumor tissues. The bladder tumor wasclinically diagnosed as transitional cell carcinoma (TCC). A summary ofthe DEK protein expression in all these bladder tissues are provided inTable 1. Note that DEK protein expression was detected in both low-gradeTCC and high-grade TCC. DEK protein was not detectable in the adjacentnormal bladder tissues, indicating high specificity. Our polyclonalanti-DEK antibody was employed in some Western blot analysis and we haveconfirmed a similar DEK protein expression in these bladder tumortissues.

TABLE 1 DEK Protein Expression in Bladder Tumor Tissues DEK ProteinExpression Tissue Cancer TCC Normal Adjacent Samples Staging GradeTissues Tissues 17135773 High T2 + − 2691046 High T2 ++++++ − 30516215High T2 Squamous − − Differentiation 44713717 High T2 ++++ − 52203387High T1 ++++++ − 65416026 High T1 ++ − A00309103 High T3a +++ − B0087901High TX ++++ − B01712101 High Tx − − E00061103 High T1 − − E00397102High ++ − E00749102 High T3 ++ − 17093709 Low TA +++ − 308936616 Low TA++++ − 313066334 Low T1 ++++++ − 314593377 Low TA ++++ − 42070185 Low TA++++++ − 53976239 Low T1 ++++++ − 60093812 Low T1 Squamous − −Differentiation 8875254 Low TA + − 9054503 Low TA +++ − A00050109 X T1++++ − A00491105 X T2b − − A00903104 X T2a ++++ − E00019101 X TX + −E0028719 X T4 ++ − E00300105 X T1 ++++ − 42012815 X Inflammation − −

In sum, we have developed a Western blot assay for detecting DEK proteinexpression. Using this assay, we have found DEK protein expression inbladder tissues from individuals suffering from low-grade and high-gradebladder cancer. The data further show that DEK protein can be found topresent in bladder tissues as early as stage 0a (i.e., Ta) in bladdercancer. Note that DEK protein is not expressed in normal healthytissues, indicating high specificity.

DEK Isoforms and Antibody Recognition—So far, we have used two (2)anti-DEK antibodies in the Western blot analysis for bladder cancer cellextracts and tissue extracts. The first antibody was a monoclonalanti-DEK antibody (cat. no. 610948) obtained from BD Bioscience (SanJose, Calif.). This monoclonal antibody was raised using syntheticpeptides corresponding to the amino acid residues 19-169 of the DEKisoform 1 (See, FIG. 4). The second antibody was a polyclonal anti-DEKantibody (cat. no. A-301-335A) available from Bethyl Labs (Montgomery,Tex.). This polyclonal antibody was raised using synthetic peptidescorresponding to amino acid residues 325-375 of the DEK isoform 1 (See,FIG. 4). DEK protein is known to encompass two (2) isoforms (namely; DEKisoform 1 and DEK isoform 2). DEK isoform 2 differs from DEK isoform 1by missing the amino acid residues 49-82. It is noted that ourmonoclonal antibody can recognize DEK isoform 1 (but not DEK isoform 2),while our polyclonal antibody can recognize both DEK isoforms. (See,FIG. 4).

We observed DEK protein expression in both bladder cancer cell lineextracts and bladder tumor tissue extracts. Because our monoclonalanti-DEK antibody can only recognize DEK isoform 1 but not isoform 2 andour polyclonal anti-DEK antibody recognizes both isoforms, we concludedthat bladder cancer cell lines and bladder tumor tissues express bothDEK isoform 1 and DEK isoform 2.

Example 4 Western Blot Detection of DEK Protein in Urine

Urine samples (in aliquots of 25 ml) were collected in the presence ofvarious protease inhibitors (e.g., aprotinin, pepstatin,phenylmethanesulfonyl fluoride, chymostatin, etc) at a concentrationsufficient to inhibit protease activity (e.g., 1 mg/ml) to avoidpotential DEK protein degradation. In this particular study, we used aprotease inhibitor cocktail (Roche, Indianapolis, Ind.). Urine samplescould be used immediately after collection or may be stored at −80° C.For the sake of convenience, most of our studies employed frozen urinesamples. Prior to Western blot analysis, frozen urine samples werethawed by leaving the samples at room temperature for 1-2 hours.

We examined if our Western blot assay could detect DEK protein in humanurine. We obtained urine samples from four (4) patients suffering frombladder cancer and one (1) healthy patient.

a) Urine Pellet

It is possible that bladder cancer cells slough off from the bladderlining into urine. To determine this possibility, we obtained urinepellet (containing potential bladder cancer cells). To do so, wecentrifuged the thawed urine (i.e., 5,000 rpm for 5 min.) to obtain theurine pellets. The urine pellets were re-suspended in 1 ml of ice coldPBS. Urine pellets were solubilized by lysing the pellets in 40 μl ofLysis Buffer B (i.e., 50 mM Tris (pH 7.4), 250 mM NaCl, 0.5% NP-40, 1%Triton X-100). Urine pellet lysates were further incubated on ice for anadditional 10 minutes. The lysates samples were centrifuged at 12,000rpm for 10 minutes. Total protein in the urine lysates was quantifiedusing a BCA assay kit (Pierce, Thermo Fisher Scientific, Rockford, Ill.)and protein concentration for each sample was adjusted to a range of0.5-1.5 μg/μl. 30 μl of urine pellet lysate was analyzed for DEK proteinexpression in solubilized urine pellets using our Western blot assaywith the polyclonal anti-DEK antibody (cat. no. A-301-335A) (detailed inExample 1). 10 μg of UroTSA cell lysate served as a control. 5 μl of theurine pellet lysate corresponding to 5 μg protein was resolved on 10%SDS-PAGE gel and stained with Coomassie blue.

FIG. 5 shows that DEK protein was not detected in the urine pellets ofthe patients suffering from bladder cancer or in the urine pellets ofhealthy patients using our Western blot assay. No protein was detectedin the urine pellets of four (4) of the five (5) samples. (See, FIG. 6).This data suggests that DEK protein cannot be detected from the urinepellets by our Western blot assay.

b) Urine Supernatant

To determine if DEK protein may be secreted or released into urine, wetested neat urine supernatant for DEK protein expression using ourWestern blot assay. In this study, urine was obtained from two (2)patients suffering from transitional cell carcinoma (TCC) and from two(2) healthy subjects.

50 μl of neat urine supernatant from each of the two (2) patientssuffering from TCC and the two (2) healthy patients was run in a Westernblot assay using our polyclonal anti-DEK antibodies (cat. no.A-301-335A). 10 μg of cell lysate from T-24 bladder cancer cells servedas a control.

FIG. 7 shows that DEK protein was not detected in the neat urinesupernatant of the two (2) patients suffering from TCC as well as fromthe two (2) healthy individuals.

To verify if there were indeed proteins present in the urinesupernatants, we resolve total proteins on a 10% SDS-PAGE gel followedby staining with Coomassie blue.

FIG. 8 clearly shows that there were abundant proteins present in eachof the neat urine supernatants tested. Thus, we concluded that DEKprotein could not be detected in neat urine supernatants from bladdercancer patients using Western blot assay.

Example 5 Western Blot Detection of DEK Protein in Concentrated Urine(by Filtration Method)

It is plausible that the neat urine may contain DEK protein that is insmall amounts beyond the sensitivity of detection by our Western blotassay. To enhance DEK protein concentration in urine samples, weconcentrated urine samples 10-fold using filtration method.

Urine samples from three (3) patients were used in this concentrationstudy: (i) a patient suffering with bladder cancer (i.e., TCC), (ii) apatient with a history of prostate cancer (i.e., HxCAP), and (iii) apatient with a history of renal cell carcinoma (i.e., HxRCC).

500 μl of the thawed urine sample was concentrated 10-fold (i.e., to afinal volume of 50 μl) using a Microcon® 3K filter (Millipore,Billerica, Mass.) (10,000 rpm, 10 min. at room temp.). All of the 50 μlof the 10-fold concentrated urine sample was analyzed for DEK proteinexpression on a Western blot assay, using the polyclonal anti-DEKantibody (cat. no. A-301-335A). UroTSA whole cell lysate (10 μg) servedas a control.

FIG. 9 shows that DEK protein was not detectable by Western blot assayeven following 10-fold concentration of the urine samples by filtrationmethod. This suggests that concentrating urine (e.g., by 10-fold) usingfiltration does not permit detecting DEK protein expression by ourWestern blot assay.

Example 6 Western Blot Detection of DEK Protein in Concentrated Urine(Chemical-Induced Precipitation)

We employed a different method to concentrate urine samples. In thisstudy, we performed a chemical-induced precipitation method. Acetone wasused as a chemical compound to cause protein precipitation in urine.Single acetone precipitation on urine samples was performed.

Potential coarse debris present in the thawed urine (25 ml) was removedby passing the thawed urine through a Kimwipe® (Kimberly-Clarke Corp.,Irving, Tex.). To induce precipitation, ice-cold acetone (volume tovolume ratio of acetone to urine was 2.5:1) was added to the urinesample. Chemical-induced precipitation was permitted to occur byincubating the acetone-treated urine at −20° C. for 1 hour.

Acetone-induced precipitates were obtained by a brief centrifugation ofthe acetone-treated urine (12,000 rpm, 10 min). The precipitates werere-suspended in a buffer containing sucrose (sucrose buffer) (i.e., 10mM triethanol amine containing 250 mM sucrose) in 500 μL volume. Thisvolume of sucrose buffer was found to be effective in re-suspending theprecipitates to solution (i.e., completely dissolve the residues).Therefore, the acetone-induced precipitation caused the urine proteinsto increase to a 50-fold concentration.

We obtained urine samples from four (4) different patient groups: (i) apatient with renal cell carcinoma (i.e., RCC); (ii) a patient withprostate cancer (i.e., CAP), (iii) a patient with benign enlargedprostate (i.e., BPH); and, (iv) a patient with transitional cellcarcinoma (i.e., TCC).

The total proteins in the sucrose-buffer re-suspended urine precipitateswere quantified using a BCA assay kit (Pierce, Thermo Fisher Scientific,Rockford, Ill.). 50 μg of protein (i.e., 50-80 μl) was used to run on aWestern blot assay using our polyclonal anti-DEK antibody (cat. no.A-301-335A). UroTSA whole cell lysate (10 μg) served as a control.

FIG. 10 shows that DEK protein was not detected by our Western blotassay in the concentrated urine samples (e.g., 50-fold concentratedurine by acetone precipitation).

In parallel, 20 μg of acetone-precipitated urine proteins were resolvedon a 10% SDS-PAGE gel and stained with Coomassie stain to test for thepresence of protein in the samples.

FIG. 11 clearly shows that proteins were present in each of the testedconcentrated urine samples. This result indicates that acetone-inducedconcentration of urine proteins (i.e., by 50-fold) is not sufficient topermit detection of DEK protein expression by our Western blot assay.

Example 7 Western Blot Detection of DEK Protein in ConsecutivelyChemical-Induced Precipitation of Urine

We further assessed if multiple chemical-induced precipitations (e.g.,acetone) would permit detection of DEK protein expression by our Westernblot assay. We repeated the same experiment as detailed above in Example6. After the single acetone precipitation, and re-suspension of theprecipitates in 500 μL of sucrose buffer.

10 ml PBS was added to the re-suspended precipitates to form PBSsolution prior to performing a second acetone precipitation. To the 10ml PBS solution we added a 2.5× volume of acetone (i.e., 25 ml ice-coldacetone) (vol/vol of acetone to PBS solution was 2.5:1).

The final pellet was re-suspended in 600 μl of sucrose buffer (10 mMTriethanolamine and 250 mM Sucrose). The amount of protein in eachsample was quantified using a BCA assay kit (Pierce, Thermo FisherScientific, Rockford, Ill.). 50 μg of protein (50-80 μl) was run on aWestern blot assay.

We tested ˜100 μg total protein of the single acetone precipitatedsamples and 100 μg total proteins from the double acetone precipitatedsamples by Western blot analysis using polyclonal anti-DEK antibody.UroTSA cell lysate (10 μg) served as a control.

FIG. 12 demonstrates that DEK protein was not detected in any of thetested samples, whether single or double acetone precipitated. Proteinswere, however, detected in all samples run on a 10% SDS-PAGE gel andstained with Coomassie blue (See, FIG. 13). This suggests that multiplechemical-induced precipitations of urine samples do not permit detectionof DEK protein by Western blot assay.

Example 8 Western Blot Detection of DEK Protein in Concentrated Urineafter Filtration-Induced Concentration Followed by Chemical-InducedPrecipitation

In this example, we examined if a combination of filtration-inducedconcentration method and chemical-induced precipitation method wouldfurther lead to concentration of urine samples and thus would permit DEKprotein detection by our Western blot assay.

In this series of study, we concentrated urine samples by: (i) firstsubjecting the urine samples to filtration-induced concentrationprotocol (i.e., concentrating urine samples with a 30K Amicon® column),and (ii) then subjecting the filter-concentrated urine samples to achemical-induced precipitation (i.e., a single acetone precipitation).

Urine samples from two (2) transitional cell carcinoma (i.e., TCC)patients were used in this study. 15 ml urine was filtered with a 30KAmicon® column by spinning the column at 6,000 rpm for 30 min. Thisfiltration-induced concentration method caused the urine sample toundergo a 30-fold increase in concentration (i.e., from 15 ml to 500μl). The 500 μl samples were re-suspended in 10 ml phosphate buffersaline (PBS).

The 10 ml re-suspended solution was then treated with ice-cold acetone(i.e., 2.5 volume of acetone was added to the solution to causeprecipitation) (See, acetone-induced precipitation protocols as detailedin Example 6). The chemical-induced precipitation further resulted in anadditional 20-fold increase in concentration. Protein was quantifiedusing BCA assay.

50 μg (˜90 μL) was analyzed for the presence of DEK protein in ourWestern blot assay using the polyclonal anti-DEK antibody (cat. no.A-301-335A). UroTSA cell lysate (10 μg) was used as a control. FIG. 14shows that DEK protein was not detected by our Western blot assay ineither of the two TCC samples.

20 μg of each sample was run on a 10% SDS-PAGE gel to test for thepresence of protein in the samples. Proteins were detected in thesamples as shown by the Coomassie blue stained 10% SDS-PAGE gel. (See,FIG. 15). This data indicates that concentrating urine by firstfiltration-induced method followed by chemical-induced method (i.e.,acetone precipitation) still does not permit DEK protein detection byWestern blot assay (despite a 600-fold increase in protein concentrationof urine).

Example 9 Western Blot Detection of DEK Protein in Concentrated Urineafter Filtration-Induced Concentration Followed by Three ConsecutiveChemical-Induced Precipitations

In this example, we examined if a combination of filtration-inducedconcentration method and consecutive chemical-induced precipitations mayfurther concentrate urine samples and thus permit detection of DEKprotein expression in our Western blot assay.

In this experiment, urine samples were: (i) first concentrated byfiltration protocol (i.e., concentrating urine samples with a 30KAmicon® column), and (ii) then concentrated by a consecutive (3×)chemical-induced precipitations (i.e., acetone precipitation).Specifically, urine sample (15 ml) from one (1) patient with bladdercancer (i.e., TCC) was concentrated with a 30K Amicon® column (spinningat 6,000 rpm for 30 min.). This filtration-induced concentration methodcaused the urine sample to undergo a 100-fold increase in concentration(i.e., from 15 ml to 150 μl). The 100-fold concentrated urine sample wasdiluted with 10 ml PBS to form a solution (PBS solution).

Then, the PBS solution (containing the concentrated urine sample afterthe filtration step) was then subjected to three (3) consecutivechemical-induced precipitations (i.e., acetone precipitation).Specifically, ice-cold acetone (volume to volume of acetone toconcentrated urine sample was 4:1) was added to the samples to causeprecipitation (i.e., adding 40 ml acetone to the 10 ml PBS solution).The mixture was chilled for an additional one (1) hour at −20° C. Themixture was centrifuged (12,000 rpm for 5 min.) to collect theprecipitates. The resulting precipitates were re-suspended in 500 μl ofbuffer A (50 mM Tris (pH 7.4), 0.5% NP-40, 1% Triton X-100). Note thatbuffer A contains a low salt content (preferably <100 mM NaCl) because ahigh salt concentration (e.g., >250 mM NaCl) is shown to adverselyaffect protein migration in a Western blot gel.

The acetone precipitation was repeated (i.e., a total of three times).Each time, the precipitates were re-suspended in 10 mL PBS prior toacetone addition. The final acetone precipitates were re-suspended in 50μL of buffer A. The present combination of filtration-inducedconcentration method and consecutive 3× chemical-induced precipitationsresulted in an increase of concentration of a total of 300-fold.

50 μL of the sample was analyzed in our Western blot assay using thepolyclonal anti-DEK antibody. FIG. 16 shows that DEK protein was stillnot detected by our Western blot assay, despite a 300-fold increase inconcentration. Similarly, no DEK protein was detected in the urinepellet (FIG. 16).

Example 10 Western Blot Detection of DEK Protein in Concentrated Urineafter Chemical-Induced Precipitation Followed by Filtration-InducedConcentration

In this example, we examined if the sequence of the concentrationprotocols may permit the detection of DEK protein expression in ourWestern blot assay.

To test this theory, we employed urine samples from seven (7) patientssuffering from transitional cell carcinoma (i.e., TCC), six (6) patientssuffering from prostate cancer (i.e., CAP), five (5) patients sufferingfrom renal cell carcinoma (i.e., RCC) and one (1) patient with a historyof transitional cell carcinoma (i.e., HxTCC).

Contrary to the sequence order of the concentration protocols detailedin Example 9, the urine samples were: (i) first concentrated by achemical-induced precipitation method (i.e., a single acetoneprecipitation), and (ii) then concentrated the urine samples by afiltration method (i.e., concentrating urine samples with a 3K Microcon®filter.

Specifically, 20 ml of urine was treated with two (2) volumes (i.e., 40mL) of ice-cold acetone. Samples were chilled for an additional one (1)hour at −20° C. and centrifuged briefly (12,000 rpm for 10 min.) tocollect the precipitates (i.e., to obtain a pellet). The pellet wasre-suspended in 2 mL of sucrose buffer (i.e., 10 mM Tri ethanol amineand 250 mM sucrose). This acetone-induced precipitation caused anincrease in concentration of urine proteins of 10-fold (i.e., from 20 mLto 2 mL).

400 μL of the re-suspended urine sample was further concentrated using a3K Microcon® filter. Samples were spun at 10,000 rpm for 10 minutes toobtain a final volume of ˜100 μL (i.e., an additional 4-fold increase inconcentration). Therefore, the combination of acetone-inducedprecipitation followed by filtration caused a total of 40-fold increasein concentration of urine. 30 μL of concentrated urine sample wasanalyzed by our Western blot assay using polyclonal anti-DEK antibody.

FIG. 17 shows DEK protein expression in the concentrated urine samples.DEK protein expression was clearly detected in four (4) of the seven (7)TCC patient samples (i.e., lanes 7, 8, 9, 18), in one (1) of the five(5) RCC patient samples (i.e., lane 14), and in one (1) of the six (6)CAP patient samples (i.e., lane 12). DEK protein was not detected in thepatient with a history of TCC (i.e., lane 16).

These results were unexpected and surprising. And they represent thefirst report that DEK protein could be detected in urine samples ofbladder cancer patients using a Western blot assay. The success of DEKprotein detection resides on the unique sequence of urine concentrations(i.e., first by a chemical-induced precipitation method followed by afiltration method).

Example 11 Patient Study—Western Blot Detection of DEK ProteinExpression in Urine from Bladder Cancer Patients

Using the sequential concentration protocols detailed in Example 10, wenext analyzed DEK protein expression in bladder cancer patient urinesamples. This study is aimed to provide a correlation between urine DEKprotein expression and the development of bladder cancer in humans.

Urine was collected from eight (8) patient groups: (i) fourteen (14)patients with varying grades of transitional cell carcinoma (i.e., TCC),(ii) eight (8) patients with prostate cancer (i.e., CAP), (iii) four (4)patients with renal cancer (i.e., RCC), (iv) three (3) nonmalignanturologenital disease (including one (1) patient suffering from cystitisand (2) suffering from chronic inflammation), (v) one (1) patientsuffering from renal oncocytoma, (vi) one (1) patient suffering fromrenal cystic nephroma, (vii) one (1) patient that had undergone radicalcystemectomy (no tumor found), and (viii) five (5) healthy individuals.

A total of thirty-six (36) urine samples (20 ml each) from the eight (8)patient groups were evaluated. Urine was sequentially concentrated usingthe acetone-precipitation method followed by the filtration method asdescribed in Example 10 (above). The concentrated urine was thenanalyzed using our Western blot assay with the polyclonal anti-DEKantibody.

Table 2 summarizes the DEK protein expression in our Western blot assay.In brief, we detected DEK protein expression in the urine of fifteen(15) of the nineteen (19) bladder cancer patients (i.e., TCC). DEKprotein expression was not detected in the urine of healthy individuals.In addition, DEK protein expression was not detected in the urine oftwelve (12) of the sixteen (16) individuals that were suffering from anon-bladder cancer ailment (e.g., CAP, RCC, chronic inflammation,cystitis).

TABLE 2 DEK Protein in Urine Samples from Bladder Cancer Patients UrineSample Source DEK Western Blot TCC Ta low grade (I) positive TCC Ta lowgrade (I-II) positive TCC Ta low grade (I) with squamous differentiationpositive TCC Ta low grade (II) negative TCC Ta low grade (I) positiveTCC Ta low grade (I) positive TCC Ta low grade (I-II) positive TCC Talow grade (I) with squamous differentiation positive TCC Ta low grade(I) positive TCC Ta low grade (I) negative TCC T1 low grade negative TCCT1 high grade with papillary + solid pattern positive TCC T1 high grade(III) + CIS with solid pattern positive TCC T1 high grade positive TCCT2 high grade + CIS positive TCC T2 high grade positive TCC T2 highgrade (III) positive TCC T2 high grade (III) & papillary RCC type1negative TCC T2 high grade (III) positive TCC inconclusive pathologynegative Chronic inflammation positive (history of TCC) No tumor onradical cystectomy negative (history of TCC) CAP negative CAP positiveCAP negative CAP negative CAP negative CAP negative CAP negative RCCnegative RCC negative RCC positive RCC positive Renal oncocytomanegative Renal cystic nephroma negative Cystitis negative Chronicinflammation negative Healthy negative Healthy negative Healthy negativeHealthy negative Healthy negative Healthy negative

FIG. 18 shows the graphical representation of DEK Western blot resultspresented in Table 2.

Altogether, this data demonstrates that DEK protein is present in theurine of bladder cancer patients. It is detected in both low grade andhigh grade bladder cancer patients. DEK protein is not found either inthe urine of healthy individuals or in the urine of non-bladder cancerpatients, indicating specificity of DEK protein as a biomarker forbladder cancer.

Overall, the present method of detecting DEK protein in urine has a highsensitivity (79%) and specificity (83%). The presence of DEK protein inurine is a viable method for detecting and diagnosing bladder cancer inhumans.

Example 12 Conductivity of Urine Samples

In this series of studies, we examined the potential mechanistic basisfor whether filtration or acetone-induced precipitation of urine maychange the salt concentrations in the urine. It is our contention thatan alteration of salt concentrations in urine may affect the outcome ofDEK protein detection in our Western blot analysis (e.g., imparting aconformational change in DEK protein).

To do so, we measured the conductivity of the urine before and after thefiltration and acetone-induced precipitation. It has been establishedthat conductivity of urine is an indication of salt concentrations(e.g., high conductivity means a high salt concentration and lowconductivity means a low salt concentration).

We processed urine samples as described in Examples 4b, 5, 6, 7, 8, 9,10 and 11. 50 μL of each sample was added to 5 mL of de-ionized water.Conductivity was determined using a conductivity meter (TraceableExpanded-Range Conductivity Meter, VWR, model no. 89094-958).Conductivity results are summarized in Table 3.

TABLE 3 Conductivity Values for Urine Samples Processing MethodsConductivity (μS/cm) None 132 (Example 4b: Neat Urine) Microcon ® 3KFiltration 146 (Example 5) Single Acetone Precipitation 110 (Example 6)Consecutive Acetone Precipitations 185 (Example 7) 30K Amicon ®Filtration followed by a 114.9 Single Acetone Precipitation (Example 8)30K Amicon ® Filtration followed by 27.6 Three Consecutive AcetonePrecipitations (Example 9) Acetone Precipitation followed by 170.6Microcon ® 3K Filtration (Example 10)

Conductivity in urine samples was decreased after acetone precipitation(1×) (i.e., Example 6), after filtration with a 30K Amicon® columnfollowed by a single acetone precipitation (i.e., Example 8), and afterfiltration followed by a consecutive (3×) acetone precipitations (i.e.,Example 9).

Conductivity in urine samples was increased after filtration with aMicrocon® 3K column (i.e., Example 5), after consecutive (3×) acetoneprecipitation (i.e., Example 7), and after acetone precipitationfollowed by filtration (i.e., Example 10).

Because DEK protein is only found to be present in the urine afteracetone-induced precipitation followed by filtration, the conductivitydata (in Table 3) does not provide a mechanistic basis for our finding.There is no clear pattern or trend of urine conductivity betweenfiltration and acetone-induced precipitation.

Example 13 Expression and Purification of Recombinant DEK Protein in theBacterial BL-21 Cells

In this series of study, we sought to recombinantly express the DEKisoform 1 and develop a capture ELISA for specific detection of DEKisoform 2.

a) cDNA and Construction of Expression Plasmid

DEK cDNA was cloned and expressed using the Gateway® system(Invitrogen). Briefly, the cDNA of the wild-type DEK isoform 1 wasobtained by the PCR amplification and cloned into pENTR™5′ TOPO® vector(Invitrogen; Carlsbad Calif.) (See Experimental Methods & Protocols).The nucleotide sequence for DEK isoform 1 cDNA was verified by DNAsequencing.

pENTR-DEK was recombined with DEST® vector (pBAD-DEST®) by LRrecombinase to obtain a DEK as a V-5 tagged protein upon expression. Thenucleotide sequence for DEK was again verified by sequencing.

b) Bacterial Expression of Recombinant DEK Isoform 1 Protein

pBAD-DEST®-DEK was transformed into BL-21 bacterial cells. DEK isoform 1protein was expressed upon induction of transformed BL-21 cells with0.2%L-arabinose at stationary phase. Protein expression was confirmed byWestern blot analysis of induced cells using a V-5 antibody and DEKmonoclonal antibody.

c) Purification of Recombinant DEK-V5 Protein from the Bacterial Cells

500 mL culture of BL-21 cells transformed with pBAD-DEST®-DEK wasinduced with 0.2% L-arabinose at stationary phase of bacterial growthfor 2 hours at 30° C. Cells were pelleted and re-suspended in BugBuster™Protein Extraction Reagent (Novagen) and sonicated. Bacterial cellextract was loaded on a Nickel-Column and DEK-V5-his was eluted using250 mM imidazole. Fractions were loaded on 10% SDS-PAGE gel and stainedwith Coomassie-blue (See, FIG. 19A) and subjected to Western blotanalysis using a V-5 monoclonal antibody (Invitrogen) (See, FIG. 19B).

Example 14 ELISA Assay to Detect DEK

To determine whether the DEK protein is present in urine of patientssuffering from bladder cancer, we developed an ELISA assay to detectDEK. In the following series of studies, we examined if DEK proteincould be detected in the urine of patients with transitional cellcarcinoma of the bladder. In order to do so, we first developed acapture and indirect ELISA using healthy urine samples that were spikedwith various concentration of recombinant DEK isoform 1 protein.

a) ELISA Development

Using the recombinant DEK isoform 1 proteins prepared (detailed above),we proceeded to develop an ELISA sandwich system. The ELISA systemallows detection of the DEK protein. In this ELISA system, two anti-DEKantibodies are used, each antibody recognizing different epitopes on DEKprotein.

b) Capture Antibody and Detection Antibody

In one of our ELISA assay, a rabbit anti-human DEK polyclonal antibody(16448-1-AP, ProteinTech Group, Chicago, Ill.) was used as a captureantibody (the polyclonal antibody was raised against full-length DEK).And, a mouse anti-human DEK monoclonal antibody (the monoclonal antibodywas raised against the amino acids 19-169 of the DEK protein) was usedas a detection antibody.

c) ELISA Sandwich

FIG. 20 depicts an ELISA sandwich using two (2) different anti-human DEKantibodies. In this series of study, the rabbit anti-human DEKpolyclonal antibody (16448-1-AP) was used as capture antibody and wascoated on the solid support (e.g., microtiter plates) in order tocapture DEK protein in spiked urine samples (i.e., control urine samplesthat were spiked with various concentrations of recombinant DEK isoform1 protein. The captured DEK was then detected by a mouse anti-human DEKmonoclonal antibody (BD Biosciences).

d) Detection System

The antibody-antigen sandwich was detected by an anti-mouse IgGconjugated with horseradish peroxidase (HRP), which specificallyrecognize mouse IgG. The peroxidase activity (representing the level ofDEK captured onto plates) was measured by addition of atetramethylbenzidine (TMB) substrate. The color intensity was directlyproportion to the amount of the bound DEK protein. Color development wasstopped by adding 1 M H₂SO₄ and the intensity of the color was measuredat optical density (OD) 450 nm using a microtiter plate reader.

e) Validation of Capture ELISA Using Recombinant DEK Isoform 1 Protein

FIG. 20 depicts the ELISA that was developed. Recombinant DEK isoform 1protein was spiked in healthy urine sample. The developed ELISA detectsthe DEK protein in the spiked urine sample.

f) Validation of Capture ELISA Using Cell Lysates from Bladder TumorTissue

FIG. 21 depicts detection of DEK protein in 50 μg of cell lysates thatwere prepared from bladder tumor tissues. In contrast, DEK protein wasnot detected in cell lysates obtained from adjacent normal bladdertissues from a TCC patient, indicating that the developed ELISA canspecifically detects DEK in human bladder tumor tissues.

Example 15 ELISA Fails to Detect DEK in Urine Samples after AcetonePrecipitation

So far, we have successfully detected DEK proteins in urines spiked withrecombinant EK isoform 1 protein and in cell lysates obtained from humanbladder tumor tissues using the developed capture ELISA. As clearlyshown in Examples 10 and 11, our Western blot assay detected DEK proteinin urines obtained from patients suffering from bladder cancer. In thesestudies detailed in Examples 10 and 11, urine samples were subjected toacetone precipitation followed by filtration-induced concentration.

To our surprise, when employed the developed capture DEK ELISA, we couldnot detect DEK protein in the urine samples obtained from the same TCCpatients. (See, FIG. 22). In contrast to the ELISA, DEK is detectableusing our Western blot assay. We reasoned that the discrepancy mayrelate to the hypothesis that ELISA is sensitive to protein thatundergoes denaturation. It is believed that DEK may undergo denaturationafter acetone precipitation.

Example 16 Acetone Precipitation is Inhibitory for Capture ELISA

We spiked urine samples with different concentrations of recombinant DEKisoform 1 protein and proceeded to concentrate the spiked urine sampleswith our established concentration steps (See, Examples 10 and 11). Inbrief, spiked urine samples were subjected to acetone precipitationfollowed by Microcon concentration as described above. The concentratedurine samples were tested by capture DEK ELISA. In this study, we failedto detect DEK protein in acetone processed urine samples, even atconcentration of 200 μg of spiked DEK protein. Noted that the developedELISA is able to detect 0.5 μg of recombinant DEK protein when spikedinto urine (i.e., without acetone treatment) (See, FIG. 23). Thus, thesedata indicate that the acetone precipitation step affects the ability ofELISA assay to detect DEK protein.

Example 17 Test Clinical Samples Using Capture ELISA by ConcentratingUrine Samples (20 Fold) Using a 3K Microcon Column

1 mL of urine (from a bladder cancer patient) was concentrated to 50 μLusing a Microcon® 3K filter and tested for DEK presence using ourdeveloped capture ELISA. As shown in FIG. 24, we could not detect DEKprotein in the urine of bladder cancer patients using the capture ELISA.It is speculated that the concentrated urine has too low amounts of DEKprotein that is below the detection limit of the capture DEK ELISA. Itis possible that DEK protein in the urine of bladder cancer patients maybe a different isoform of DEK protein that may not be detectable by themouse monoclonal DEK antibody used in the capture ELISA.

Example 18 Urine DEK Protein Contains DEK Isoform 2

DEK proteins are present in two isoforms; namely, DEK isoform 1 and DEKisoform 2. The detection antibody used in the capture ELISA onlyrecognizes DEK isoform 1 and not DEK isoform 2. It is therefore possiblethat patients with TCC have DEK isoform 2 solely in the urine. Thereforewe speculated that an anti-DEK antibody that would detect DEK isoform 2would detect DEK in the urine of bladder cancer patients.

In this study, we compared the ability of three (3) different anti-DEKantibodies in detecting urine DEK from patients with bladder cancer. Inthis study, we used (i) a polyclonal anti-DEK antibody (#16448-1-AP,raised against the full length DEK isoform 1); (ii) a monoclonalanti-DEK antibody (#610948, raised against amino acids 19-169 DEKisoform 1); and (iii) a polyclonal anti-DEK antibody (A301-335A, raisedagainst amino acids 325-375).

2 ml of urine sample from patients with bladder cancer and from healthyindividual was concentrated to 100 μL (20 fold concentration) using a 3KMicrocon® Filter and coated on a microtiter plate. The three (3)different antibodies were compared in its ability to detect DEK in urineof clinical samples. The antigen-antibody complex was detected byanti-mouse or anti-rabbit IgG conjugated with horseradish peroxidase(HRP). The peroxidase activity (representing the level of DEK) wasmeasured by addition of tetramethylbenzidine (TMB) substrate. The colorintensity was in direct proportion to the amount of the bound DEK. Colordevelopment was stopped by adding 1 M H₂SO₄ and the intensity of thecolor was measured at optical density (OD) 450 nm on a microtiter platereader.

Results were summarized in Table 4. The two (2) polyclonal anti-DEKantibodies were able to detect urine DEK. In contrast, the monoclonalanti-DEK antibody failed to detect DEK. This data is consistent with ourobservation that the capture ELISA could not detect DEK in any of theclinical urine samples. Hence, any two (2) anti-DEK antibodies(recognizing different sites on DEK protein) could be used to detect DEKisoform 2 in the capture ELISA format.

TABLE 4 Comparison of Detection of Urine DEK by Indirect ELISARecognizes Recognizes Detect DEK in DEK DEK Urine of Bladder Isoform 1Isoform 2 Cancer Patients Monoclonal Yes No No DEK Ab (#610948)Polyclonal Yes Yes Yes DEK Ab (#A301-335A) Polyclonal Yes Yes Yes DEK Ab(#16448-1-AP)

Table 5 summaries the detection of DEK using a polyclonal DEK antibody(325-375) and a monoclonal DEK antibody (19-169) in detecting DEKprotein from various biological samples.

TABLE 5 Ability of DEK Antibody in Detecting DEK Protein in VariousBiological Sources DEK protein detection DEK protein DEK protein inbladder tumor detection in in urine from tissues and cultured bladdertumor bladder cancer bladder cancer cells tissues patients MonoclonalDEK Yes Yes No Antibody (#610948) Polyclonal DEK Yes Yes Yes Antibody(#A301-335A)

Example 19 Development of Indirect DEK ELISA for Detection of DEKProtein in Urine Samples

Based on the results in Example 18, we developed an indirect ELISA fordetecting DEK protein in urines of bladder cancer patients using ananti-DEK polyclonal antibody (#A301-335A).

Urine samples (2 ml aliquots) from (i) patients suffering bladder cancer(various grades), (ii) patients suspected TCC, (iii) patients withhistory of TCC and (iv) healthy individuals were concentrated to 100 μl(i.e., 20 fold concentrated) using a 3K Microcon filter. Concentratedurines were directly coated on microtiter plates.

An anti-DEK polyclonal antibody (A301-335A) was used for detection ofbound DEK protein on the wells of the microtiter plates. Theantigen-antibody complex was detected by anti-rabbit IgG conjugated withhorseradish peroxidase (HRP). The peroxidase activity (representing thelevel of DEK) was measured by addition of tetramethylbenzidine (TMB)substrate. The color intensity was in direct proportion to the amount ofthe bound DEK.

DEK protein was detected in TCC urine samples, but not in the urine ofhealthy donors and patient with history of TCC (See, FIG. 25). Thissuggests that using an indirect ELISA method we were able to detect DEKprotein specifically in the urine of bladder cancer patients.

Example 20 Limit of Detection for DEK Indirect ELISA

We determined the limit of detection of urine DEK using the indirectELISA. For standard curve, we spiked 2 mL of urine samples with variousconcentration of recombinant DEK isoform 1 protein and concentrated thespiked urine samples to 20 fold and coated on the wells of microtiterplates. Next, we processed urine samples obtained from 25 healthyindividual and employed the DEK polyclonal antibody (A301-335A), and theindirect ELISA as described in experiment 19. Based on standard curve ofDEK, we determined the cut-off value for positive detection of DEK inurine samples at a concentration greater than 1.5 μg of DEK protein in20-fold concentrated urine samples (See, FIG. 26).

Example 21 Correlation Between Urine DEK and Bladder Cancer

Next, we tested urine samples from 41 patients with TCC, healthy donors,renal cell carcinoma, history of TCC, non-malignant disease andindividual with suspected bladder cancer. We used an indirect DEK ELISAand successfully detected DEK protein in urines from bladder cancerpatients. The indirect DEK ELISA assay has a sensitivity of 67% andspecificity of 85% (See, FIG. 27). This suggests that there is a strongcorrelation between the presence of DEK protein (isoform 2) in urines ofpatients with bladder cancer. Table 6 summarizes the data presented inFIG. 27.

TABLE 6 DEK Detection in Clinical Samples using Indirect ELISA DEK inUrine TCC H. TCC NMD RCC Suspected Tumor Healthy Positive 15 0 1 1 1 1Negative 8 2 0 3 0 9

Example 22 Limit of Detection of Polyclonal Anti-DEK Antibody(A301-335A) by Western Blot and ELISA

We determined the limit of detection of DEK polyclonal antibody byWestern blot and by indirect ELISA. We spiked urine samples obtainedfrom healthy individual and with increasing concentration of recombinantDEK isoform 1.

For Western blot assay, 13 ml of urine was spiked with 2.5, 5, 10, 15,20, 30, 50, 60, 75 μg/mL concentration of recombinant DEK isoform 1.Spiked urine sample was subjected to acetone precipitation. Pellet wasre-suspended in 2 mL of sucrose buffer. 400 μL of re-suspended bufferwas concentrated using a 3K Microcon filter. Results indicate that atconcentration of 15 μg/ml and higher DEK was detected in urine byWestern blot using the DEK polyclonal antibody (A301-335A).

For indirect ELISA, we spiked 2, 4, 6, 8, 10, 16, 20 μg in 2 mL of urinesamples. Spiked urine samples were concentrated using a 10K Microconfilter to a final volume of 125 μL. 125 μL was coated on the wells andsubjected to indirect ELISA. Results indicate that concentration of ≧2μg/mL recombinant DEK could be detected by indirect ELISA.

Table 7 summarizes the limit of detection study comparing Western blotanalysis and ELISA assay.

TABLE 7 Limit of Detection - Western Blot v. Indirect ELISA Limit ofDetection of Recombinant Methods DEK in Spiked Urine Samples WesternBlot Assay 15 μg/mL Indirect ELISA Assay  2 μg/mL

Example 23 Production of Monoclonal DEK Antibodies Raised AgainstPeptide Sequences Corresponding to DEK Isoform 2

In this experiment, we sought to generate several monoclonal antibodiesagainst human DEK protein. Our goal is employ the generated monoclonalantibodies in an ELISA. We used synthetic peptides as immunogens toraise monoclonal antibodies in mice. To ensure a better outcome, weprepared long synthetic peptide sequences (>30 amino acids) and shortsynthetic peptide sequences (<15 amino acids) that correspond to humanDEK isoform 2 protein sequence (NCBI Accession No. for human DEK proteinisoform 2 is NP_001128181.1) (SEQ ID NO: 2) and used them as immunogensfor the generation of the monoclonal antibodies. FIG. 28 shows thelocation of corresponding peptide sequence in DEK isoform 2 for thegeneration of monoclonal antibodies.

In the initial testing, we used Western blot analysis and indirect ELISAto test the bleeds from mice injected with the short or long DEK isoform2 peptides using recombinant His-tagged DEK protein (rDEK-His). Based onthe data obtained from the Western blot and antigen down ELISA, onlythree (mAb 16, mAb 260 and mAb 302) out of the four test bleeds derivedfrom mice injected with long peptides indicated an immune responseagainst DEK. The test bleeds derived from the mice injected with shortpeptides did not have an immune response against DEK. The reason for thefailure in immune response and mAb production is unclear. Table 8summarizes the reactivity of the peptide sequences against the DEKprotein based on Western blot analysis and antigen down ELISA of testbleeds. In sum, using six (6) peptide sequences as immunogens, wesuccessfully prepared mAb 16, mAb 260 and mAb 302.

TABLE 8  Peptides for Monoclonal Antibody Production andAnalysis of Test Bleeds for Immunogenicity Amino Acid WB Residues of Analysis DEK and DEK Isoform 2 Indirect Monoclonal Used for ELISAAntibody Generating of Test ID mAb Bleeds Peptide SequenceLONG PEPTIDES (>30 amino acids) mAb 16 aa 16-50 √CQPASEKEPEMPGPREESEEEEDEDDEEEEEEEKGK (SEQ ID NO: 4) mAb 260 aa 260-300 √QNSSKKESESEDSSDDEPLIKKLKKPPTDEELKETIKKLLAC (SEQ ID NO: 5) mAb 207aa 207-240 Produced  EESSDDEDKESEEEPPKKTAKREKPKQKATSKSKC No(SEQ ID NO: 6) Clones mAb 302 aa302-341 √CSANLEEVTMKQICKKVYENYPTYDLTERKDFIKTTVKELIS (SEQ ID NO: 7)SHORT PEPTIDES (<15 amino acids) mAb 159 aa 159-173 Produced CLPKSKKTCSKGSKK No (SEQ ID NO: 8) Clones mAb 173 aa 173-187 Produced CERNSSGMARKAKRT No (SEQ ID NO: 9) Clones

Example 24 Analysis of Purified Monoclonal DEK Antibodies by WesternBlot

We next used the mice that showed an immune response against DEK formaking the hybridomas. The mice corresponding to mAb16, mAb260 andmAb302 were used for cell fusion. After cell fusion (hybridomas), weestablished multiple clones for the three (3) mAbs. Specifically, weprepared three (3) single clones for mAb16 (namely, mAb 16-1D4F8, mAb16-1D4 F10 and mAb 16-2C9C3), three (3) single clones for mAb260(namely, mAb 260-6C5G8, mAb 260-6F9F6 and mAb 260-6F9F6) and two (2)single clones for mAb 302 (namely, mAb 302-2B9A8 and mAb 302-3E9E11).

Next, we performed a Western blot analysis on the purified (isolated)monoclonal antibodies from the corresponding hybridomas using the 5637cell line expressing DEK Sh RNA (DEK knockdown), non-specific ShRNA andagainst rDEK-His. We preformed a Western blot analysis of purifiedmonoclonal DEK antibody on cell lysate and rDEK His protein (FIG. 29).The purified antibodies were only able to detect DEK in the 5637 cellline with nonspecific ShRNA, as such the antibodies are specific for theDEK protein (Table 9).

Example 25 Analysis of Purified Monoclonal DEK Antibodies by IndirectELISA Using Recombinant DEK Spiked into PBS

In this series of experiments, we tested purified (isolated) monoclonalantibodies from the corresponding clones using an antigen down ELISA. Wespiked various concentrations of rDEK into PBS. We spiked rDEK at 1μg/mL and prepared a two-fold dilution series, such that our finalconcentrations were 1 μg/mL, 0.5 μg/mL. 0.25 μg/mL, 0.125 μg/mL, 0.062μg/mL, 0.031 μg/mL and 0.015 μg/mL. 300 μL of sample was used in theantigen down ELISA and 300 μL of PBS was used as blank (i.e., controlwithout rDEK). We used the purified antibodies at dilutions ranging from1:1000 to 1:8000. We show that the purified antibodies tested in theantigen down ELISA were able to detect rDEK in PBS at a concentration of15 ng/ml or less as shown in Table 9.

Example 26 Analysis of Purified Monoclonal DEK Antibodies by IndirectELISA Using Recombinant DEK Spiked into Urine

In this series of studies, we tested whether the purified monoclonalantibodies were able to detect rDEK-His when spiked into neat urine. Wespiked various concentrations of rDEK into the neat urine. We spikedrDEK at 1 μg/mL and prepared a two-fold dilution series, such that ourfinal concentrations of rDEK were 1 μg/mL, 0.5 μg/ml, 0.25 μg/mL, 0.125μg/mL, 0.062 μg/mL, 0.031 μg/mL and 0.015 μg/mL. 300 μL of spiked neaturine was used in the antigen down ELISA and 300 μL of neat urine wasused as blank. The purified antibodies were used at dilutions rangingfrom 1:1000 to 1:8000.

We show that the purified monoclonal antibodies were able to detect rDEKin neat urine (Table 9). However, the sensitivity of detection variedbetween the different antibodies as well as between the different clones(Table 9).

TABLE 9 Summary of Purified (Isolated) Antibodies Tested by Western Blotand by Antigen Down ELISA Using PBS or Urine Western Blot AnalysisIndirect ELISA Specifics Recombinant PBS Urine Monoclonal Cell DEK Limitof Limit of Antibody ID Lysate protein Dilution Detection DilutionDetection mAb 16-1D4F8 ✓ ✓ 1:1000-1:8000 0.015 μg/mL 1:1000-1:80000.125-0.062 μg/mL mAb 16-1D4F10 ✓ ✓ 1:1000-1:8000 0.015 μg/mL1:1000-1:8000 0.25-0.125 μg/mL mAb 16-2C9C3 ✓ ✓ 1:1000-1:8000 0.015μg/mL 1:2000-1:8000 0.125-0.062 μg/mL mAb 260-6C5G8 ✓ ✓ 1:1000-1:80000.015 μg/mL  1:500-1:8000 0.25-0.125 μg/mL mAb 260-6D11F2 ✓ ✓1:1000-1:8000 0.015 μg/mL  1:500-1:4000 0.015 μg/mL mAb 260-6F9F6 ✓ ✓1:1000-1:8000 0.015 μg/mL  1:500-1:2000 0.015 μg/mL mAb 302-2B9A8 ✓ ✓1:1000-1:8000 0.015 μg/mL  1:500-1:2000 0.015-0.007 μg/mL mAb 302-3E9E11✓ ✓ 1:1000-1:8000 0.015 μg/mL  1:500-1:2000 0.015-0.007 μg/mL

Example 27 Sandwich ELISA Using New Monoclonal DEK Antibodies andRecombinant DEK Spiked in Urine

We tested different combinations of the newly raised monoclonal DEKantibodies in a sandwich ELISA using recombinant DEK spiked in neaturine obtained from a healthy donor. We immobilized a capture antibodyon an ELISA plate and added various dilutions (ranging from 250 ng/mL to2 ng/mL concentrations) of recombinant DEK (rDEK) spiked in urine todetermine the limit of detection. 300 μL of neat spiked urine was testedin the sandwich ELISA. The detection monoclonal antibody wasbiotinylated using PEG₁₂ biotin and HRP and the labeled strepavidinantibody was used for detection of the sandwich complex (Table 10).

After multiple experiments, we found that only the pair consisting ofmAb16-2C9C3 as the capture antibody and mAb260-6F9F6 and the detectionantibody was able to detect DEK at a low concentration of 7-4 ng/mL inneat urine. All other tested pairs of monoclonal antibodies were onlyable to detect high concentrations (>250 ng/mL to 15 ng/mL) in neaturine. Concentrations of less than 10 ng/mL were considered the limit ofdetection for a sensitive sandwich ELISA to detect DEK.

TABLE 10 Summary of Limit of Detection of Various Pairs of DEKMonoclonal Antibodies Using Recombinant DEK Spiked in Urine DetectionAntibody mAb 260- mAb 16- mAb 302- mAd 302- Capture 6F9F6 2C9C3 2B9A83E9E11 Antibody (biotin) (biotin) (biotin) (biotin) mAb 260- N/A* (same62-31 62-31 62-31 6F9F6 recognition ng/mL ng/mL ng/mL sites) mAb 16-7.8-4 N/A (same >2 15-7.8 2C9C3 ng/mL recognition μg/mL (High ng/mLsites) Background) mAb 302- 15-7.8 >250 N/A (same N/A (same 2B9A8 ng/mLng/mL recognition recognition sites) sites) mAb 302- 15-7.8 >250 N/A(same N/A (same 3E9E11 ng/mL ng/mL recognition recognition sites) sites)N/A* means not available. ELISA cannot be performed when same antibodyis used.

Example 28 Performance of DEK Sandwich ELISA (mAb 16-2C9C3 and mAb260-6F9F6-Biotin) in DEK Protein Spiked PBS and Urine

We analyzed the performance of the DEK sandwich ELISA, consisting of mAb16-2C9C3 as the capture antibody and mAb 260-6F9F6-biotin as thedetection antibody, in detecting DEK protein spiked in either PBS orneat urine. We prepared rDEK standards in both PBS and urine. For urine,we used rDEK with a starting concentration of 125 ng/mL followed by atwo-fold dilution series such that the lowest concentration of rDEK was1.9 ng/mL. For PBS, we used rDEK with a starting concentration of 15.6ng/ml followed by a twofold dilution series such that the lowestconcentration of rDEK is PBS was 0.24 ng/mL, 300 μl of spiked PBS orspiked neat urine.

Our results indicate that the DEK sandwich ELISA detected rDEK at aconcentration of ˜4 ng/mL (FIG. 30A) whereas in PBS there was only a 2fold increase in sensitivity to ˜2 ng/mL (FIG. 30B) indicating that theantibody pair (mAb 16-2C9C3 and mAb 260-6F9F6) used in the DEK sandwichELISA has minimum interfering effects of urine with high sensitivity andtherefore can be used on neat urine for the detection of DEK (FIG. 29A).We have successfully developed a highly sensitive ELISA for DEK usingthe monoclonal antibodies (mAb 16-2C9C3 and mAb 260-6F9F6) that permitthe detection of DEK at as low as 4 ng/ml in neat urine. To oursurprise, the use of this anti-DEK monoclonal antibody pairs requiresthe ELISA without the use of concentrated urine. In other words, incontrast to the ELISA described in Example 17, neat urine is permittedwhen using mAb 16-2C9C3 and mAb 260-6F9F6 as the monoclonal antibodypair. Our new much improved DEK sandwich ELISA with high sensitivity isdepicted in FIG. 31.

In further studies, we sequenced the clones 2C9C3 (mAb 16-2CC3) and6F9F6 (mAb 260-6F9F6). FIG. 32 shows the location of the different CDRwithin the heavy and light chain of these monoclonal antibodies.

Example 29 DEK Sandwich ELISA (mAb 16-2C9C3 and mAb 260-6F9F6-Biotin)for the Detection of DEK

The capture antibody (mAb 16-2C9C3) was coated on a 96 well ELISA plateat a concentration of 1-2 μg/mL in bicarbonate-carbonate buffer. Next,the plate was incubated at 4° C. for 1 hour. The plate was washed withwash buffer (1×PBS containing 0.05% Tween 20) and Casein blocking bufferis added to the well and incubated on a 96 well plate shaker at 450 rpmfor 1 hour. Next, 300 μL of neat urine (clinical sample) and 300 μL ofDEK (various rDEK protein dilutions prepared in synthetic urine) wasadded to the respective wells followed by incubation for 2 hours at roomtemperature. Following the washing step, the biotinylated detection DEKantibody mAb 260-6F9F6-biotin was added at a concentration of 1-2 μg/mLin 1×PBS and incubated for 1 hour. Following a wash step, HRP linkedStrepavidin antibody was then added at 1:100 dilution and incubated for1 hour. 150 μL of HRP substrate was then added to the washed wells andcolor development is allowed to take place for 2-10 min. The plate wasthen read at 450 and 590 nm.

Example 30 Performance of DEK Sandwich ELISA (mAb 16-2C9C3 andmAb260-6F9F6-Biotin) on Clinical Urine Sample to Detect Bladder Cancer

We previously published that the DEK protein is elevated in the urine ofbladder cancer patients. We tested our unexpectedly sensitive DEKsandwich ELISA on 35 clinical urine samples from patients suffering frombladder cancer (16 patients) and from patients with non-bladder cancerurogenital diseases (cystitis, stone and renal cell carcinoma) andhealthy patients (19 patient samples).

Our DEK sandwich ELISA has a sensitivity of 82.3% and a specificity of70.58%. The positive predictive value (PPV) of the sandwich ELISA is 75%and the negative predictive value (NPV) is 84.6% (FIG. 33).

Example 31 Performance Characteristics of the Improved DEK SandwichELISA Using mAb 16-2C9C3 and mAb 260-6F9F6 (Biotin)

We sought to determine the performance characteristics of our DEKsandwich ELISA comprising of mAb 16-2C9C3 as the capture antibody andmAb 260-6F9F6-biotin as the detection antibody. We prepared syntheticurine as a control to determine the performance characteristics. We madesynthetic urine that consisted of 0.9% NaCl and 2% human albumin inwater. We used the DEK sandwich ELISA to test rDEK spiked in syntheticurine and spiked healthy urine to confirm the synthetic urine as acontrol. We determined that assay sensitivity in synthetic urine wassame as healthy urine samples (FIG. 34).

The following characteristics of the DEK sandwich ELISA were determinedand are tabulated in Table 11:

I. Assay Sensitivity:

i) Assay sensitivity was determined by loading 10 sets of blanks(synthetic urine) as unknown samples and determining the result of themean, plus 3 standard deviations (SD) of the 10 mean results, on thestandard curve. Known concentration rDEK were run in duplicates for thestandard curve.Assay Sensitivity=Mean of blank+3SD of blank

Based on the above equation the assay sensitivity of DEK sandwich ELISAwas determined to be 3.9 ng/ml.

ii) We calculated the limit of blank from the 10 sets of blank which wasused for limit of detection calculations.

-   -   Limit of Blank (LoB):        LoB=mean blank+1.645(SD blank)        II. Limit of Detection (LoD):

Limit of detection is the lowest concentration of DEK protein that canbe measured reliably with the DEK sandwich ELISA. Based on the ClinicalLaboratory standard Institute EP17 2004 publication, LoD was determinedby utilizing both the measured limit LoB and test replicates of a sampleknown to contain a low concentration of analyte.

Six (6) replicates of all the different concentration of rDEK used inthe standard curve were run in the DEK sandwich ELISA. The mean andstandard deviation of the lowest concentration of the standards wascalculated according and LoD is determined as indicated below.LoD=LoB+1.645 (SD low concentration sample)

The SD of the lowest concentration calibrator should be less than 5% ofLoB LoD was determined to be 3.9 ng/mL.

III. Limit of Quantification (LoQ)

We ran 10 sets of blanks (healthy urine only) as unknown samples anddetermined the result of the mean plus 10 times the standard deviations(SD) of the 10 mean results, on the standard curve.

Limit of quantification defined here as reagent blank+10 times standarddeviation of reagent blank. We then ran the standards in duplicate forthe standard curve.LoQ=Mean blank+10×SD of blankIV. Linearity of Dilution:

At least 5 (five) urine samples (clinical urine sample from bladdercancer patients) containing DEK were serially diluted with syntheticurine and assayed in duplicates. Linear regression analysis of the DEKantigen concentration versus dilution was performed.

Linearity of reportable range was performed carrying out serialdilutions of a known positive in synthetic urine then comparing theplotted regression value. The plotted regression value should not exceed10% variance.

Panel of Analytes: (the Diluent Solution: Synthetic Urine))

A=the positive control neat

B=1:2 dilution of the positive control

C=1:4 dilution of the positive control

D=1:8 dilution of the positive control

E=1:16 dilution of the positive control

F=the negative control neat

Each dilution was run in duplicate. The average value is plotted againstthe dilution factor. The slopes for the samples should range between0.8-1.05 with a correlation coefficient of greater than 0.9, thusdemonstrating that the samples will dilute linearly

V. Recovery

At least 3 positive samples were used to determine if DEK in urine canbe recovered, suggesting that the DEK sandwich ELISA is specific for theDEK antigen. Known concentration of DEK from patient urine was added tonegative urine sample at a 1:1 dilution. The samples were measured induplicate. The mean of 2 assays is reported. Mean recoveries of DEKantigen from positive patient sample range between 84-107%.

VI. Precision

Precision testing is necessary to assess the reproducibility of theassay. For example, DEK positive urine samples must be consistentlypositive respectively to prove that there is true DEK detection whenmultiple operators test identical samples. Conversely, DEK negativesamples must also consistently test negative when multiple operatorsrepeat the assay. Precision testing was performed by running at least 3known positive urine samples and 3 known negative urine sample intriplicates by 3 different operators on 3 separate days. The OD valuesshould not deviate more than 15%. Reproducibility analysis of theprecision panel was performed upon completion of all challenges.Reproducibility was translated into Inter-Assay Standard Derivation (SD)and Coefficient of Variation (CV %). Coefficient of Variation (CV %) wasranged from 7-30%.

TABLE 11 Performance Characteristics of DEK sandwich ELISA AssaySensitivity 3.9 ng/mL Limit of Detection 3.9 ng/mL Limit ofQuantification 7.8-15 ng/mL   Linearity Correlation Coefficient0.91-0.97 Precision % CV 7-30% Recovery 84-107%

Experimental Methods and Procedures

1. Cell Lines: Human bladder cancer cell lines (e.g., T-24 and RT-4)were maintained in McCoy's 5A medium supplemented with 10% fetal bovineserum (FBS). Human bladder cancer cell lines (e.g., 5637 and TCCSUP)were maintained in RPMI supplemented with 10% FBS. SV-40 transformedhuman bladder urothelium cell line (i.e., UroTSA cell line) wasmaintained in DMEM medium supplemented with 10% FBS. Human bladderepithelium progenitor cell line (i.e., HBEP) (obtained from CELL N TEC®,Stauffacherstr, Bern, Switzerland) was maintained in CnT-58 medium.Differentiated epithelial cells were maintained in accordance with themanufacturer's protocol. All cell lines were maintained at 37° C. in 5%CO₂.

2. Whole Cell Lysates from Cell Lines: Prior to cell lysis, culturedcells were washed with 10 ml of cold PBS. Cells were lysed using 1 mL ofRIPA buffer (150 mM NaCl, 0.01 M sodium pyrophosphate, 10 mM EDTA, 10 mMsodium fluoride, 50 mM Tris ph 8.8, 0.1% SDS, 12.8 mM deoxycholic acid,10% glycerol, 1% NP-40) supplemented with protease inhibitors (Roche,Indianapolis, Ind.) at a concentration of 1 μg/μL. Lysed cells werescraped and transferred to 1.5 ml centrifuge tube and centrifuged at14,000 rpm for 10 min. to collect supernatant (i.e., whole celllysates).

3. Urine and Tissue Sample Collections: Urine samples were obtained fromconsented patients with bladder cancer (i.e., TCC), prostate cancer(i.e., CAP), renal cancer (i.e., RCC), non-malignant urogenital diseasesand healthy individuals. Urine samples (˜20-50 ml aliquots) werecollected from patients in Wolfson Medical Center (Israel). Urinesamples were stored in the presence of protease inhibitors (CompleteProtease Inhibitor Tablets, Roche, Indianapolis, Ind.) at 1 μg/μL. Urinesamples were immediately stored at −20° C. and shipped on dry ice. Uponarrival, samples were stored at −80° C.

Frozen, cold cut tissue samples from bladder tumor and adjacent normaltissues were obtained from patients from Wolfson Medical Center (Israel)as well as from ABS Analytical Biological Services Inc. (US).

4. Tissue Extracts from Tissue Samples: Cold cut tissue samples werecollected and immediately frozen upon removal. Samples were shipped ondried ice and stored at −80° C. To obtain tissue extract, cold cutfrozen tissue samples were homogenized in RIPA buffer (400 μl) using amortar and pestle. Homogenized tissue was centrifuged at 14,000 rpm at4° C. for 20 min and supernatant was saved for downstream analysis. Theamount of protein in each sample was quantified using a BCA assay kit(Pierce, Thermo Fisher Scientific, Rockford, Ill.).

5. Protein Extraction from Urine Pellet: Urine was centrifuged at 3,000rpm for 5 min. Pellet was washed three times with 10 ml PBS. Washedurine pellet was lysed in 100 μL lysis buffer (150 mM NaCl, 0.2%TritonX-100 and 10 mM Tris pH 7.4). 50 μL of urine pellet lysate wasused for Western or Coomassie-blue staining studies.

6. Urine Pellet Lysates: To obtain lysates from urine pellet, urine wasfirst centrifuged at 3,000 rpm for 5 minutes to obtain a pellet. Thepellet was then washed three times with 10 mL PBS and then lysed in 100μl lysis buffer B (150 mM NaCl, 0.2% TritonX-100 and 10 mM Tris pH 7.4).50 μL of the urine pellet lysate was used in the Western blot assay orin protein detection gels.

7. Chemical-Induced Precipitation of Protein in Urine: Fresh urinesamples were used in chemical induced protein precipitation.Alternatively, frozen urine samples were thawed at room temperatureprior to protein precipitation. Chemicals used in protein precipitationincluded acetone, ethanol, TCA, and methanol-chloroform. These chemicalswere used (preferably maintained at −20° C. when in use) in an amountsufficient to induce formation of precipitates in urine. Theprecipitates were then re-suspended in a sucrose buffer (10 mMtriethanolamine and 250 mM sucrose).

8. Preparation of DEK Knockdown Cell Lines: 293FT cells (cat. no.R700-07, Invitrogen, Carlsbad, Calif.) were used in the preparation ofDEK-ShRNA lentivirus particles. A DEK shRNA(TGCTGTTGACAGTGAGCGCGCACATTTGGCTTACAGTAAATAGTGAAGCCACAGATGTATTTACTGTAAGCCAAATGTGCTT GCCTACTGCCTCGGA)(SEQ ID NO: 1) was used to knockdown DEK expression. A lentiviral vectorcontaining the DEK shRNA (pGIPZ-DEK shRNA) (cat. no. RHS4430-99137795)was purchased from Open Biosystems (ThermoScientific, Huntsville, Ala.).To prepare DEK-ShRNA lentivirus, 5×10⁵ 293 FT cells were firsttransfected with 10 μg of pGIPZ-DEK shRNA and 5 μg of the packagingvectors (i.e., pCMVΔR8.2 and pHCMV-G) (a gift from Dr. Lairmore, TheOhio State University) (Wei, et al., Journal of Virology, February 2006,p1242-1249, Vol. 80, No. 3) and grown at 37° C. in 5% CO₂. Supernatantsof the transfected cells (containing lentivirus particles) werecollected at 24 and 48 hours post-transfection. Debris in thesupernatants was removed using a 0.45 μm filter.

To obtain DEK knockdown UroTSA cell line, 10⁸ UroTSA cells wastransduced with DEK shRNA lentiviral particles, 5 ml of thevirus-containing filtrate was spread onto a 10 mm tissue culture dishcontaining confluent UroTSA cells. Cells were selected for DEK shRNAexpression at 48 hours using puromycin (2.5 μg/μL) (Sigma-Aldrich, St.Louis, Mo.).

9. Preparation of DEK Over-Expressing Cell Lines: DEK gene (NCBIAccession No. NM_003472.3) was cloned into a lentiviral vector(GATEWAY®). To prepare DEK over-expressing cells, 5×10⁵ 293 FT cellswere first transfected with the DEK-lentiviral vector pLenti6/V5-DESTGateway® (cat. no. K4960-00, Invitrogen, Carlsbad, Calif.) and apackaging mix (ViraPower™ BSD Packaging Mix, cat. no. K490-00,Invitrogen, Carlsbad, Calif.). Supernatants from the transfected cellswere collected at 24 and 48 hours post-transfection and passed through a0.45 μm filter at 50,000 rpm for 2 hours to remove debris.

5 ml of the virus-containing filtrate was spread onto a 10 mm tissueculture dish containing confluent UroTSA cells. Cells were selected forDEK-V5 expression at 48 hours using blasticidin (10 μg/μL) (Invitrogen,Carlsbad, Calif.).

10. Conductivity Measurements: Various concentrations (0.1 mM. 0.25 mM,0.5 mM, 2.5 mM, 5 mM and 10 mM) of potassium chloride were used tocalibrate the conductivity meter (Traceable Expanded-Range ConductivityMeter, VWR, model no. 89094-958). Samples (e.g., urine) were prepared byadding 50 μl of sample to 5 ml of de-ionized water. Conductivity wasmeasured by inserting the conductivity probe into the samples and isexpressed as μs/cm.

11. Preparation of DEK Knockdown and DEK-V5 Expressing UroTSA and 5637Cell Lines

DEK shRNA in lentiviral vector (pGIPZ) was purchased from OpenBiosystems. Co-transfected 293FT cells with DEK shRNA pGIPZ, packagingvectors 8.2 and Env plasmids. 24 hours and 48 hours post-transfectionculture supernatants containing the packaged lentiviral particles wascollected and passed through a 0.45 μm filter. UroTSA and 5637 celllines were transduced using the DEK shRNA containing lentivirus. 48hours post transduction, cells expressing DEK shRNA was selected usingpuromycin. UroTSA DEK-V5 stable cell line was obtained by cloning DEK inGATEWAY® lentiviral vector and co-transfected with packaging mix fromInvitrogen. Virus was harvested and cells transduced as described aboveand stable cell line expressing DEK-V5 selected using blasticidin.

12. Production of DEK Monoclonal Antibodies

Peptide was conjugated with KLH as immunogen and 5 BALB/c mice wereimmunized. The mice with satisfied immune response were used for cellfusion and hybridoma production. Briefly, the spleen of mice withsatisfied immune response was taken and B cells were isolated from thesplenocytes. Next the B cells were fused with Sp2/o murine myeloma cellsand selected on HAT (hyoxanthine aminopterine thymidine) media.Surviving hybridomas were serially diluted into multi-well plates tosuch an extent that each well contained only one cell. The single cellwas termed as a clone, since the antibodies in a well were produced bythe same B cell, and was directed towards the same epitope. Supernatantsof clones for corresponding initial peptide sequence were screened byindirect ELISA and the clone which showed maximum reactivity towards DEKantigen was used for the production of antibody. Five (5) mouse ascitesproduction were done for 1 selected cell line (Clone) and the producedmonoclonal antibodies in the ascites were purified by Protein G affinitycolumn. The monoclonal antibodies obtained were determined to be IgG1isotype.

13. Biotinylation of Monoclonal Antibody

EZ-Link NHS-PEG12Biotin (#21312) was purchased from Thermo Scientific(Rockford, Ill.) and manufacturer's protocol was followed. We dissolved1-10 mg protein to be modified in PBS. We removed an appropriate volumeof 250 mM Biotin Reagent Stock Solution based on calculations using a 5-to 20-fold molar excess of EZ-Link NHS-PEG12-Biotin for proteinsolutions >2 mg/mL, dispensed it into the protein solution and mixedwell. We then incubated the reaction on ice for two hours, removedexcess non-reacted and hydrolyzed biotin reagent using a desaltingcolumn.

All publications and patents cited in this specification are hereinincorporated by reference in their entirety. Although the invention hasbeen described in connection with specific preferred embodiments andcertain working examples, it should be understood that the invention asclaimed should not be unduly limited to such specific embodiments.Various modifications and variations of the described composition,method, and systems of the invention will be apparent to those skilledin the art without departing from the scope and spirit of the invention.

What is claimed is:
 1. A method of detecting DEK protein in a biologicalsample, comprising the steps of: a. providing a biological sample; b.immobilizing an isolated monoclonal antibody (mAb 16-2C9C3) onto a solidsurface, c. said mAb 16-2C9C3 comprises: (i) CDR1 of the heavy chainvariable region which has the amino acid sequence of SEQ ID NO:15; (ii)CDR2 of the heavy chain variable region which has the amino acidsequence of SEQ ID NO:16; (iii) CDR3 of the heavy chain variable regionwhich has the amino acid sequence of SEQ ID NO:17; (iv) CDR1 of thelight chain variable region which has an amino acid sequence of SEQ IDNO: 23; (v) CDR2 of the light chain variable region which has the aminoacid sequence of SEQ ID NO:24; and (vi) CDR3 of the light chain variableregion which has the amino acid sequence of SEQ ID NO:25; d. adding saidbiological sample onto said solid surface to allow DEK protein presentin said biological sample to be captured onto said solid surface by saidisolated monoclonal antibody (mAb 16-2C9C3); e. washing the solidsurface to remove unbound DEK protein; f. adding an isolated monoclonalantibody (mAb 260-6F9F6) so as to allow formation of a complex betweensaid captured DEK protein with said isolated monoclonal antibody (mAb260-6F9F6), said mAb 260-6F9F6 comprises: (i) CDR1 of the heavy chainvariable region which has the amino acid sequence of SEQ ID NO:31; (ii)CDR2 of the heavy chain variable region which has the amino acidsequence of SEQ ID NO:32; (iii) CDR3 of the heavy chain variable regionwhich has the amino acid sequence of SEQ ID NO:33; (iv) CDR1 of thelight chain variable region which has an amino acid sequence of SEQ IDNO: 39; (v) CDR2 of the light chain variable region which has the aminoacid sequence of SEQ ID NO:40; and (vi) CDR3 of the light chain variableregion which has the amino acid sequence of SEQ ID NO:41; and g.detecting said complex, wherein the presence of said complex isindicative of the presence of DEK in said biological sample; and whereinsaid biological sample is urine.
 2. A method of detecting DEK protein ina biological sample, comprising the steps of: a. providing a biologicalsample; b. immobilizing an isolated monoclonal antibody (mAb 16-2C9C3)onto a solid surface, said mAb 16-2C9C3 comprises a heavy chain variableregion having the amino acid sequence of SEQ ID NO: 14 and a light chainvariable region having the amino acid sequence of SEQ ID NO: 22; c.adding said biological sample onto said solid surface to allow DEKprotein present in said biological sample to be captured onto said solidsurface by said isolated monoclonal antibody (mAb 16-2C9C3); d. washingthe solid surface to remove unbound DEK protein; e. adding an isolatedmonoclonal antibody (mAb 260-6F9F6) so as to allow formation of acomplex between said captured DEK protein with said isolated monoclonalantibody (mAb 260-6F9F6), said mAb 260-6F9F6 comprises a heavy chainvariable region having the amino acid sequence of SEQ ID NO: 30 and alight chain variable region having the amino acid sequence of SEQ ID NO:38; and f. detecting said complex, wherein the presence of said complexis indicative of the presence of DEK in said biological sample; andwherein said biological sample is urine.